Liquid pharmaceutical composition

ABSTRACT

The present invention relates to novel liquid pharmaceutical compositions of adalimumab, which include adalimumab or a biosimilar thereof, an histidine buffering agent such as histidine (or histidine buffer system such as histidine/imidiazolium-histidine), and a sugar stabiliser such as trehalose. Such a combination of components furnishes formulations having a stability (e.g. on storage and when exposed to stress) which is comparable to or an improvement upon those known in the art, and with fewer ingredients. Such advances will help adalimumab treatments to become more widely available at lower cost, and prolong the viability of pre-loaded delivery devices (e.g. pre-filled syringes) to reduce unnecessary waste of the drug.

INTRODUCTION

The present invention relates to a novel protein formulation. Inparticular, the invention relates to a liquid pharmaceutical compositionof adalimumab, to a method of manufacturing the composition, to a kitincluding the composition, to a package including the composition, to amethod of manufacturing the package, and to methods of treatment usingthe composition and/or package.

BACKGROUND

Treatment of tumour necrosis factor-alpha (TNF-α)-related autoimmunediseases, such as rheumatoid arthritis, psoriasis and other autoimmunediseases, has been achieved through the use of FDA-approved drugs suchas Adalimumab (HUMIRA®, Abbott Corporation). Adalimumab is a humanmonoclonal antibody that inhibits human TNF-α activity so as to preventit from activating TNF receptors, thereby downregulating inflammatoryresponses associated with autoimmune diseases. Approved medicalindications for Adalimumab include rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis,moderate to severe chronic psoriasis and juvenile idiopathic arthritis.

Adalimumab is generally delivered to a patient via subcutaneousinjection, and is thus provided in a liquid form, typically in packagessuch as vials, preloaded syringes, or preloaded “pen devices”.Commercially available pen devices (HUMIRA® Pen) generally include a 1mL pre-filled glass syringe, preloaded with 0.8 mL of a sterileformulation of 40 mg Adalimumab (see below), with a fixed needle (eithergray natural rubber or a latex free version) and a needle cover.Commercial formulations (HUMIRA®) of Adalimumab contain the followingingredients:

Amount per container (mg) Amount Ingredient (filling volume = 0.8 mL)(mg/mL) Adalimumab 40 50 Citric Acid Monohydrate 1.04 1.3 Dibasic sodiumphosphate 1.22 1.53 dihydrate Mannitol 9.6 12 Monobasic sodium phosphate0.69 0.86 dihydrate Polysorbate 80 0.8 1 Sodium chloride 4.93 6.16Sodium citrate 0.24 0.3 WFI and sodium hydroxide q.b. to adjust q.b. toadjust pH to 5.2 pH to 5.2

Adalimumab, and its method of manufacture, is described in WO97/29131(BASF) as D2E7, and elsewhere in the art.

Though the aforementioned commercial formulation of Adalimumab is stable(at least to some extent), the relevant antibody may be unstable overprolonged periods or under stressed conditions, thus precludingprolonged storage of said formulations. Such degradation of theformulation may be due to a variety of factors, including:

-   -   Physical effects, such as:        -   Inadequate inhibition of aggregation of the relevant protein            molecules (a function supposedly served by Tween-80);        -   Inadequate inhibition of precipitation;        -   Inadequate inhibition of adsorption of the relevant protein            molecules at the interface of water and air or at the            contact surface of any packaging material (a function            supposedly served by Tween-80);        -   Inadequate regulation of osmotic pressure (a function            supposedly served by mannitol);    -   Chemical effects, such as:        -   Inadequate regulation of oxidation (a function supposedly            served by mannitol and potentially undermined by Tween-80,            which can promoted oxidation of double bonds);        -   Inadequate inhibition of photo-oxidation;        -   Inadequate inhibition of hydrolysis of ester bonds leading            to the formation of acid, aldehyde and peroxide products,            thus affecting the stability of the antibody;        -   Inadequate stabilisation and maintenance of pH;        -   Inadequate inhibition of protein fragmentation;        -   Inadequate inhibition of protein unfolding;

Any, some, or all of the above factors can lead to either an unviabledrug product (which may be unsafe for use in medical treatments) or adrug product whose viability is variable and unpredictable, especiallyin view of the variable stresses (agitation, heat, light) differentbatches of drug product may be exposed to during manufacture, transport,and storage.

In terms of the physical and chemical stabilisation of Adalimumab, thecomplex array of components within the aforementioned commercialformulations appears to perform below expectations, especially in viewof the large number of components. Though this particular combination ofexcipients undoubtedly represents a ‘delicate balance’ (given theinterplay between various technical factors) and was the result ofextensive research and development, in view of the apparent risk ofunderperformance it is questionable whether such a large number ofdifferent excipients is justified, especially given that this inevitablyincreases processing and cost burdens, toxicity risks, and risks ofdeleterious interactions between components that could compromise theformulation. Even if the overall performance of the commercialformulations could not be surpassed, an alternative formulation havingcomparative performance but containing few components would represent ahighly desirable replacement for the commercial formulations, for atleast the aforesaid reasons.

In order to guarantee reproducible clinical performance of aprotein-based pharmaceutical product, such products must remain in astable and consistent form over time. It is well-established thatmolecular alterations can occur during every stage of the manufacturingprocess, including during the production of the final formulation andduring storage. Molecular alterations can modify a quality attribute ofa biopharmaceutical product, resulting in an undesirable change in theidentity, strength or purity of the product. Some such problems areoutlined above.

The primary goal of formulation development is to provide apharmaceutical composition that will support the stability of abiopharmaceutical protein during all stages of its production, storage,shipping and use. Formulation development for an innovativebiopharmaceutical protein, or a biosimilar monoclonal antibody (mAb), isessential to its safety, clinical efficacy and commercial success.

There is therefore a need for the provision of alternative or improvedliquid formulations of adalimumab. Desirably, any new formulations wouldsolve at least one of the aforementioned problems and/or at least oneproblem inherent in the prior art, and may suitably solve two or more ofsaid problems. Desirably, the problem(s) of the prior art may be solvedwhilst reducing the complexity of the formulation.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is provided aliquid pharmaceutical composition comprising adalimumab (which suitablyincludes any biosimilar thereof); a histidine buffering agent (orhistidine buffer system); and a sugar stabiliser; wherein thecomposition optionally comprises (or excludes) any one or moreadditional components defined herein in relation to a liquidpharmaceutical composition (e.g. including tonicifier, excludingarginine, etc.), optionally in any amount, concentration, or formstipulated herein; and wherein the composition optionally exhibits anyone or more parameters or properties given herein in relation to aliquid pharmaceutical composition (e.g. pH, osmolality, aggregation,fragmentation, protein unfolding, turbity, etc.).

According to a second aspect of the present invention there is provideda liquid pharmaceutical composition comprising adalimumab; an histidinebuffering agent (or histidine buffer system); and a sugar stabiliser;wherein the composition has a pH greater than or equal to pH 6.30.

According to a second aspect of the present invention there is provideda liquid pharmaceutical composition comprising adalimumab; an histidinebuffering agent (or histidine buffer system); and a sugar stabiliser;wherein the composition is either (substantially or entirely) free ofarginine (suitably L-arginine) or comprises arginine in a concentrationof at most 0.1 mM.

According to a fourth aspect of the present invention there is provideda liquid pharmaceutical composition comprising adalimumab; an histidinebuffering agent (or histidine buffer system); and a sugar stabiliser;wherein the composition is either (substantially or entirely) free ofamino acids other than histidine or comprises one or more amino acidsother than histidine in a (collective) concentration of at most 0.1 mM.

According to a fifth aspect of the present invention there is provided apackage (e.g. pre-filled syringe, pen, intravenous bag, or apackage/container containing any of the aforementioned) comprising aliquid pharmaceutical composition as defined herein.

According to a sixth aspect of the present invention there is provided adrug delivery device (e.g. pre-filled syringe or pen, or intravenousbag) comprising a liquid pharmaceutical composition as defined herein.

According to a seventh aspect of the present invention there is provideda kit of parts comprising a drug delivery device, a liquidpharmaceutical composition as defined herein (optionally contained in apackage or container), and optionally a set of instructions withdirections regarding the administration (e.g. sub-cutaneous) of theliquid pharmaceutical composition.

According to an eighth aspect of the present invention there is provideda method of manufacturing a liquid pharmaceutical composition, themethod comprising mixing together adalimumab; an histidine bufferingagent (or histidine buffer system); a sugar stabiliser; and optionallyany one or more additional components defined herein in relation to aliquid pharmaceutical composition, optionally in any amount,concentration, or form stipulated; and optionally adjusting any one ormore parameters given herein in relation to a liquid pharmaceuticalcomposition (e.g. pH, osmolality).

According to a ninth aspect of the present invention there is provided aliquid pharmaceutical composition obtainable by, obtained by, ordirectly obtained by a method of manufacturing a liquid pharmaceuticalcomposition as defined herein.

According to a tenth aspect of the present invention there is provided amethod of manufacturing a package or a drug delivery device, the methodcomprising incorporating a liquid pharmaceutical composition as definedherein within a package or drug delivery device.

According to an eleventh aspect of the present invention there isprovided a package or a drug delivery device obtainable by, obtained by,or directly obtained by a method of manufacturing a package or a drugdelivery device as defined herein.

According to a twelfth aspect of the present invention there is provideda method of treating a disease or medical disorder in a patient in needof such treatment, said method comprising administering to said patienta therapeutically effective amount of a liquid pharmaceuticalcomposition as defined herein.

According to a thirteenth aspect of the present invention there isprovided a liquid pharmaceutical composition as defined herein for usein therapy.

According to a fourteenth aspect of the present invention there isprovided a use of a liquid pharmaceutical composition as defined hereinin the manufacture of a medicament for the treatment of a disease ordisorder.

According to a fifteenth aspect of the present invention there isprovided a method of treating a tumour necrosis factor-alpha(TNF-α)-related autoimmune disease in a patient in need of suchtreatment, said method comprising administering to said patient atherapeutically effective amount of a liquid pharmaceutical compositionas defined herein.

According to a sixteenth aspect of the present invention there isprovided a liquid pharmaceutical composition as defined herein for usein the treatment of a tumour necrosis factor-alpha (TNF-α)-relatedautoimmune disease.

According to a seventeenth aspect of the present invention there isprovided a use of a liquid pharmaceutical composition as defined hereinin the manufacture of a medicament for the treatment of a tumournecrosis factor-alpha (TNF-α)-related autoimmune disease.

According to an eighteenth aspect of the present invention there isprovided a method of treating rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, Crohn's disease, ulcerative colitis, moderate tosevere chronic psoriasis and/or juvenile idiopathic arthritis in apatient in need of such treatment, said method comprising administeringto said patient a therapeutically effective amount of a liquidpharmaceutical composition as defined herein.

According to a ninteenth aspect of the present invention there isprovided a liquid pharmaceutical composition as defined herein for usein the treatment of rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, Crohn's disease, ulcerative colitis, moderate tosevere chronic psoriasis and/or juvenile idiopathic arthritis.

According to a twentieth aspect of the present invention there isprovided a use of a liquid pharmaceutical composition as defined hereinin the manufacture of a medicament for the treatment of rheumatoidarthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease,ulcerative colitis, moderate to severe chronic psoriasis and/or juvenileidiopathic arthritis.

In further aspects, the invention provides a liquid pharmaceuticalcomposition, a package, a drug delivery device, a kit of parts, a methodof manufacturing a liquid pharmaceutical composition, a method ofmanufacturing a package or a drug delivery device, a method of treating,a liquid pharmaceutical composition for use, and a use of a liquidpharmaceutical composition in the manufacture of a medicament,essentially as defined herein (including in any of the aforementionedtwenty aspects) except that, rather than being specific to “adalimumab”(and biosimilars thereof), the invention may instead apply (and therebybe defined as relating) to any TNF-α-inhibiting antibody (anti-TNF-αantibody) (or any biosimilar thereof), albeit suitably an antibody thatinhibits human TNF-α activity, and most suitably a human monoclonalantibody that inhibits human TNF-α activity. Suitably the anti-TNF-αantibody is a therapeutically effective medicament (at least whenadministered in appropriate quantities to a patient in need thereof) (ora biosimilar thereof—see below for definitions of biosimilars inrelation to adalimumab, which applies equally to all anti-TNF-αantibodies), suitably one which has received FDA approval. As such, anyreference herein to “adalimumab” may, unless incompatible therewith, beconstrued as a reference to any anti-TNF-α antibody for the purpose ofthese additional aspects of the invention (whether this relates toabsolute or relative amounts, concentrations, parameters, or properties,or whether it relates to certain definitions, such as what constitutes abiosimilar).

One of these further aspects of the present invention provides a liquidpharmaceutical composition comprising an anti-TNF-α antibody (whichsuitably includes any biosimilar thereof); a histidine buffering agent(or a histidine buffer system); and a sugar stabiliser; wherein thecomposition optionally comprises (or excludes) any one or moreadditional components defined herein in relation to a liquidpharmaceutical composition (e.g. including surfactant, excludingarginine, etc.), optionally in any amount, concentration, or formstipulated herein; and wherein the composition optionally exhibits anyone or more parameters or properties given herein in relation to aliquid pharmaceutical composition (e.g. pH, osmolality, aggregation,fragmentation, protein unfolding, turbity, etc.).

In a particular embodiment, the anti-TNF-α antibody is selected from thegroup including adalimumab, infliximab, certolizumab pegol, golimumab.

Any features, including optional, suitable, and preferred features,described in relation to any particular aspect of the invention may alsobe features, including optional, suitable and preferred features, of anyother aspect of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the invention, and to show how embodimentsof the same are put into effect, reference is now made, by way ofexample, to the following diagrammatic drawings, in which:

FIG. 1 is a bar chart showing the protein content (mg/mL), as determinedby OD, of the DoE1 formulations (of Example 1), along with referencestandards (representing comparator HUMIRA® formulations), at anarbitrary start point (blue bars, time=0) and after 4 weeks (red bars)of the formulation(s) being heated at 40° C.

FIG. 2 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE1 formulations (of Example 1), along with referencestandards (representing comparator HUMIRA® formulations), at anarbitrary start point (blue bars, time=0) and after both 2 weeks (greenbars) and 4 weeks (orange bars) of the formulation(s) being heated at40° C.

FIG. 3 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE1 formulations (of Example 1), along withreference standards (representing comparator HUMIRA® formulations), atan arbitrary start point (dark blue bars, time=0) and after both 2 weeks(pink bars) and 4 weeks (light blue bars) of the formulation(s) beingheated at 40° C.

FIG. 4 is a bar chart showing the unfolding temperature (° C.), asdetermined by DSF, of the DoE1 formulations (of Example 1), along withreference standards (representing comparator HUMIRA® formulations).

FIG. 5 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE2 formulations (of Example 2), along with referencestandards (representing comparator HUMIRA® formulations), at anarbitrary start point (red bars, time=0) and after both 2 weeks (greenbars) and 4 weeks (purple bars) of the formulation(s) being heated at40° C.

FIG. 6 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE2 formulations (of Example 2), along withreference standards (representing comparator HUMIRA® formulations), atan arbitrary start point (blue bars, time=0) and after both 2 weeks (redbars) and 4 weeks (green bars) of the formulation(s) being heated at 40°C.

FIG. 7 is a bar chart showing the main peak isoforms profile, asdetermined by iCE280 analysis, of the DoE2 formulations (of Example 2)at an arbitrary start point (blue bars, time=0) and after both 2 weeks(red bars) and 4 weeks (green bars) of the formulation(s) being heatedat 40° C.

FIG. 8 is a bar chart showing the acid cluster peak(s) isoforms profile,as determined by iCE280 analysis, of the DoE2 formulations (of Example2) at an arbitrary start point (blue bars, time=0) and after both 2weeks (red bars) and 4 weeks (green bars) of the formulation(s) beingheated at 40° C.

FIG. 9 is a bar chart showing the turbidity, as determined byNephelometry, of the DoE2 formulations (of Example 2) at an arbitrarystart point (blue bars, time=0) and after both 2 weeks (red bars) and 4weeks (green bars) of the formulation(s) being heated at 40° C.

FIG. 10 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE2 formulations (of Example 2) at an arbitrary startpoint (blue bars, time=0) and after both 24 hours (red bars) and 48hours (green bars) of the formulation(s) being mechanically agitated(shaking).

FIG. 11 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE2 formulations (of Example 2) at an arbitrarystart point (blue bars, time=0) and after both 24 hours (red bars) and48 hours (green bars) of the formulation(s) being mechanically agitated(shaking).

FIG. 12 is a bar chart showing the turbidity, as determined byNephelometry, of the DoE2 formulations (of Example 2) at an arbitrarystart point (blue bars, time=0) and after both 24 hours (red bars) and48 hours (green bars) of the formulation(s) being mechanically agitated(shaking).

FIG. 13 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE2 formulations (of Example 2), along with referencestandards (representing comparator HUMIRA® formulations), beforeexposure to light (blue bars, time=0) and after 7-hour light exposure at765 W/m² (red bars).

FIG. 14 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE2 formulations (of Example 2) before exposure tolight (blue bars, time=0) and after 7-hour light exposure at 765 W/m²(red bars).

FIG. 15 is a bar chart showing the main peak isoforms profile, asdetermined by iCE280 analysis, of the DoE2 formulations (of Example 2),along with reference standards (representing comparator HUMIRA®formulations), before exposure to light (blue bars, time=0) and after7-hour light exposure at 765 W/m² (red bars).

FIG. 16 is a bar chart showing the acid cluster peak(s) isoformsprofile, as determined by iCE280 analysis, of the DoE2 formulations (ofExample 2), along with reference standards (representing comparatorHUMIRA® formulations), before exposure to light (blue bars, time=0) andafter 7-hour light exposure at 765 W/m² (red bars).

FIG. 17 is a bar chart showing the turbidity, as determined byNephelometry, of the DoE2 formulations (of Example 2) before exposure tolight (blue bars, time=0) and after 7-hour light exposure at 765 W/m²(red bars).

FIG. 18 is a bar chart showing the main peak isoforms profile, asdetermined by iCE280 analysis, of the DoE2 formulations (of Example 2)before (blue bars, time=0) and after m² (red bars) five freeze-thawingcycles (−80° C.→room temperature).

FIG. 19 is a bar chart showing the acid cluster peak(s) isoformsprofile, as determined by iCE280 analysis, of the DoE2 formulations (ofExample 2) before (blue bars, time=0) and after m² (red bars) fivefreeze-thawing cycles (−80° C.→room temperature).

FIG. 20 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE2 formulations (of Example 2), along with referencestandards (representing comparator HUMIRA® formulations) before (bluebars, time=0) and after m² (red bars) five freeze-thawing cycles (−80°C.→room temperature).

FIG. 21 is a bar chart showing the number concentration (#/mg) ofsub-visible particles with a particle size less than or equal to 10microns, as determined by sub-visible particle count analysis, of theDoE2 formulations (of Example 2) before (blue bars, time=0) and after m²(red bars) five freeze-thawing cycles (−80° C.→room temperature).

FIG. 22 is a bar chart showing the number concentration (#/mg) ofsub-visible particles with a particle size less than or equal to 25microns, as determined by sub-visible particle count analysis, of theDoE2 formulations (of Example 2) before (blue bars, time=0) and after m²(red bars) five freeze-thawing cycles (−80° C.→room temperature).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise stated, the following terms used in the specificationand claims have the following meanings set out below.

References herein to “adalimumab” include the originator drug substance(as commercially available), adalimumab as defined in WO97/29131 (BASF)(particularly D2E7 therein) and elsewhere in the art, and alsobiosimilars thereof. D2E7 of WO97/29131 “has a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 3 and a heavy chainCDR3 domain comprising the amino acid sequence of SEQ ID NO: 4”.Preferably, the D2E7 antibody has a light chain variable region (LCVR)comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chainvariable region (HCVR) comprising the amino acid sequence of SEQ ID NO:2. WO97/29131 gives details of each of these sequence listings.References herein to “adalimumab” may include biosimilars which, forinstance, may share at least 75%, suitably at least 80%, suitably atleast 85%, suitably at least 90%, suitably at least 95%, suitably atleast 96%, suitably at least 97%, suitably at least 98% or most suitablyat least 99% protein sequence identity with any one of protein sequencesdisclosed in either WO97/29131 (especially in relation to D2E7) orelsewhere in relation to “adalimumab”. Alternatively or additionally,references herein to “adalimumab” may include biosimilars which exhibitat least 75%, suitably at least 80%, suitably at least 85%, suitably atleast 90%, suitably at least 95%, suitably at least 96%, suitably atleast 97%, suitably at least 98% or most suitably at least 99% proteinsequence homology with any one of protein sequences disclosed in eitherWO97/29131 (especially in relation to D2E7) or elsewhere in relation to“adalimumab”. Alternatively or additionally, a biosimilar may have a(slightly) different glycosylation profile, even if the protein sequenceis substantially the same or different to the extent specified above.

The term “biosimilar” (also know as follow-on biologics) is well knownin the art, and the skilled person would readily appreciate when a drugsubstance would be considered a biosimilar of adalimumab. Furthermore,such “biosimilars” would need to be officially approved as a“biosimilar” for marketing before said “biosimilar” is sold on the openmarket. The term “biosimilar” is generally used to describe subsequentversions (generally from a different source) of “innovatorbiopharmaceutical products” (“biologics” whose drug substance is made bya living organism or derived from a living organism or throughrecombinant DNA or controlled gene expression methodologies) that havebeen previously officially granted marketing authorization. Sincebiologics have a high degree of molecular complexity, and are generallysensitive to changes in manufacturing processes (e.g. if different celllines are used in their production), and since subsequent follow-onmanufacturers generally do not have access to the originator's molecularclone, cell bank, know-how regarding the fermentation and purificationprocess, nor to the active drug substance itself (only the innovator'scommercialized drug product), any “biosimilar” is unlikely to be exactlythe same as the innovator drug product.

For the purposes of various molar calculations (e.g. for molar ratiosbetween adalimumab and another component of the liquid pharmaceuticalcomposition of the invention) the molecular weight of adalimumab may betaken to be 144190.3 g/mol (reference molecular weight) based on detailsdisclosed on the CAS database for CAS #331731-18-1, Adalimumab, wherethe molecular formula is taken as C₆₄₂₈H₉₉₁₂N₁₆₉₄O₁₉₈₇S₄₆. As such, aliquid pharmaceutical composition containing 50 mg/mL adalimumab may beconsidered a 0.347 mM (or 347 μM) solution of adalimumab. This is notintended to be in any way limiting regarding the nature of anybiosimilars of adalimumab covered by the scope of the present invention,nor the level of glycosylation, either of which may effect the actualmolecular weight. However, where a biosimilar does have a differentmolecular weight, the abovementioned reference molecular weight shouldbe suitably used for the purposes of assessing whether or not such abiosimilar falls within the scope of any molar definitions stipulatedwithin this specification. So the number of moles in a known weight ofsaid biosimilar should be calculated, just for the purposes of thisinvention, using the above reference molecular weight.

Herein, the term “buffer” or “buffer solution” refers to a generallyaqueous solution comprising a mixture of an acid (usually a weak acid,e.g. acetic acid, citric acid, imidazolium form of histidine) and itsconjugate base (e.g. an acetate or citrate salt, for example, sodiumacetate, sodium citrate, or histidine) or alternatively a mixture of abase (usually a weak base, e.g. histidine) and its conjugate acid (e.g.protonated histidine salt). The pH of a “buffer solution” will changevery only slightly upon addition of a small quantity of strong acid orbase due to the “buffering effect” imparted by the “buffering agent”.

Herein, a “buffer system” comprises one or more buffering agent(s)and/or an acid/base conjugate(s) thereof, and more suitably comprisesone or more buffering agent(s) and an acid/base conjugate(s) thereof,and most suitably comprises one buffering agent only and an acid/baseconjugate thereof. Unless stated otherwise, any concentrationsstipulated herein in relation to a “buffer system” (i.e. a bufferconcentration) suitably refers to the combined concentration of thebuffering agent(s) and/or acid/base conjugate(s) thereof. In otherwords, concentrations stipulated herein in relation to a “buffer system”suitably refer to the combined concentration of all the relevantbuffering species (i.e. the species in dynamic equilibrium with oneanother, e.g. citrate/citric acid). As such, a given concentration of ahistidine buffer system generally relates to the combined concentrationof histidine and the imidazolium form of histidine. However, in the caseof histidine, such concentrations are usually straightforward tocalculate by reference to the input quantities of histidine or a saltthereof. The overall pH of the composition comprising the relevantbuffer system is generally a reflection of the equilibrium concentrationof each of the relevant buffering species (i.e. the balance of bufferingagent(s) to acid/base conjugate(s) thereof).

Herein, the term “buffering agent” refers to an acid or base component(usually a weak acid or weak base) of a buffer or buffer solution. Abuffering agent helps maintain the pH of a given solution at or near toa pre-determined value, and the buffering agents are generally chosen tocomplement the pre-determined value. A buffering agent is suitably asingle compound which gives rise to a desired buffering effect,especially when said buffering agent is mixed with (and suitably capableof proton exchange with) an appropriate amount (depending on thepre-determined pH desired) of its corresponding “acid/base conjugate”,or if the required amount of its corresponding “acid/base conjugate” isformed in situ—this may be achieved by adding strong acid or base untilthe required pH is reached. By way of example:

-   -   A histidine “buffering agent” is the free amino acid, histidine.        Since amino acids such as histidine are amphoteric, and thus        capable of behaving as both an acid and base, the “buffering        agent” is simply the amphoteric compound itself (suitably in        zwitterionic form). However, a histidine buffer system or buffer        solution may optionally have, added thereto in addition to        histidine, a quantity of acid (suitably a strong acid, such as        hydrochloric acid) or base (suitably a strong base, such as        sodium hydroxide) until the desired pH is reached. As such, some        of the histidine present may exhibit a different protonation        state than the zwitterionic amino acid. Herein, except where the        contrary is stated, any concentrations given in relation to a        histidine buffer system suitably refer to the combined        concentration of the buffering agent (e.g. histidine) and/or        acid/base conjugate(s) thereof (e.g. imidazolium form of        histidine). The skilled person is readily able to calculate such        concentrations, and may do so by simple reference to the input        quantities of histidine or its acid/base conjugate (e.g.        histidine hydrochloride). Such concentrations may be calculated        by reference to the combined concentrations of buffering        agent(s) and acid/base conjugate(s), where a buffer system is        formed by simply mixing together buffering agent(s) and        acid/base conjugate(s). Alternatively, where a buffer system is        formed by mixing either the buffering agent(s) or acid/base        conjugate(s) with a pH adjuster (e.g. strong acid or strong        base) to produce a mixture of each, suitably such concentrations        may be calculated by reference to the starting        amounts/concentrations of the buffering agent(s) or acid/base        conjugate(s) respectively. For example, where a buffer system is        formed using a known amount/concentration of histidine which is        mixed with a pH adjuster (e.g. sodium hydroxide) until the        desired pH is reached, the concentration of the buffer system        may be calculated by reference to the initial amount of        histidine. Likewise, the same applies where a buffer system is        formed using a known amount/concentration of histidine        imidazolium salt (e.g. histidine hydrochloride) mixed with a pH        adjuster (e.g. sodium hydroxide) until the desired pH is        reached—in this case the concentration of the buffer system may        be calculated by reference to the initial amount of histidine        imidazolium salt.

Herein, an “acid/base conjugate” refers to the conjugate acid orconjugate base (whichever is relevant at a particular pH—typically theconjugate acid in the context of the present invention) of a particular“buffering agent”. The acid/base conjugate of a histidine bufferingagent (e.g. histidine) is suitably the imidazolium form of histidine,suitably an imidazolium salt of histidine. The imidazolium form ofhistidine may be referred to herein as “imidazolium-histidine”, and hasthe structure:

An imidazolium salt of histidine may be referred to ashistidine-imidazolium salt, and has essentially the same structure asshown above save for an associated counteraction.

Herein, the term “buffering species” refers to the particular species(excluding any associated counteranions or countercations—i.e. ignorechloride or hydroxide counter-ions for histidine/imidazolium-histidinesystems) of a given buffer system which are in dynamic equilibrium with(and proton-exchange with) one another. For example, histidine andimidazolium-histidine may together constitute the “histidine bufferingspecies” of a “histidine buffer system”.

Since it is often somewhat difficult to define quantities (whetherabsolute or relative) of a buffer system by reference to weight (sincethe total weight will depend on the desired pH, which will affect theamount of counterions present), herein weight-based quantities mayinstead be determined by reference to a theoretical weight of therelevant “buffering species”. At least two species are generally presentin any given set of “buffering species” (in relative amounts that canonly be determined by reference to the pH), each with a differentmolecular weight (which usually differs by just 1). Therefore, to enableviable weight calculations and references, for the purposes of thisspecification the weight of any given set of “buffering species” isgiven as a theoretical weight based on just one of the bufferingspecies, namely the most basic of the buffering species (i.e. the leastprotonated form at any given pH). So the weight of a given set of“buffering species” is quoted as the weight of basic-speciesequivalents. By way of example, in a histidine buffer system thehistidine buffering species may consist of histidine andimidazolium-histidine cations. The weight of the “buffering species” istherefore calculated as if histidine was the only species present in thebuffer system (even if imidiazolium-histidine is present alongsidehistidine). Thus, any reference to a weight or weight ratio involving a“histidine buffering species” suitably refers to the theoretical weightof histidine equivalents within the buffer system. As such, where acomposition is formed by adding a pH adjuster (e.g. sodium hydroxide) toa fixed amount of imidazolium histidine, or indeed to a fixed amount ofhistidine (which may suitably form some imidazolium-histidine upondissolution in the diluent), the original weight of histidine may beconsidered to be the weight of the “buffering species” regardless of theultimate pH. Alternatively, if the concentration (i.e. molarity) of abuffer system is known, this can be converted into a weight of“buffering species” by reference to the molecular weight of the mostbasic form of the relevant buffering species (e.g. histidine), andignoring the fact that imidazolium-histidine cations are also present.

Unless stated otherwise, references herein to an “amino acid” or “aminoacids”, whether specific (e.g. arginine, histidine) or general (e.g. anyamino acid), in the context of their presence or otherwise withincompositions (especially pharmaceutical liquid compositions of theinvention) relate to the corresponding free amino acid(s) (regardless ofits/their protonation state and/or salt form, though for consistencyamounts are suitably calculated by reference to the free amino acid perse). This may suitably include natural and/or artificial amino acids.Unless stated to the contrary, such references are not intended torelate to amino acid residue(s) covalently incorporated as part of alarger compound (as opposed to a composition comprising multiplecompounds), such as a peptide or protein (where such amino acid residuesare linked via peptide bonds). As such, though adalimumab, as a protein,contains amino acid residues, it is not considered to comprise any “freeamino acid(s)”. By way of example, a composition defined as being “freeof arginine” does not contain any free arginine but it may still includeone or more proteins (e.g. adalimumab) which do themselves comprisearginine residues.

Unless stated otherwise, references herein to any one or more “aminoacids”, whether specific or general, suitably relate to theL-stereoisomers or a racemate thereof, most suitably L-amino acids.

The term “substantially free”, when used in relation to a givencomponent of a composition (e.g. “a liquid pharmaceutical compositionsubstantially free of arginine”), refers to a composition to whichessentially none of said component has been added. As explained above,such references have no bearing on the presence of amino acid residue(s)within a protein structure. When a composition is “substantially free”of a given component, said composition suitably comprises no more than0.001 wt % of said component, suitably no more than 0.0001 wt % of saidcomponent, suitably no more than 0.00001 wt %, suitably no more than0.000001 wt %, suitably no more than 0.0000001 wt % thereof, mostsuitably no more than 0.0001 parts per billion (by weight).

The term “entirely free”, when used in relation to a given component ofa composition (e.g. “a liquid pharmaceutical composition substantiallyfree of arginine”), refers to a composition containing none of saidcomponent. As explained above, such references have no bearing on thepresence of amino acid residue(s) within a protein structure.

Herein, in the context of the present specification, a “strong acid” issuitably one having a pK_(a) of −1.0 or less, whereas a “weak acid” issuitably one having a pK_(a) of 2.0 or more. Herein, in the context ofthe present specification, a “strong base” is suitably one whoseconjugate acid has a pK_(a) of 12 or higher (suitably 14 or higher),whereas a “weak base” is suitably one whose conjugate acid has a pK_(a)of 10 or less.

Herein, a “stabiliser” refers to a component which facilitatesmaintenance of the structural integrity of the biopharmaceutical drug,particularly during freezing and/or lyophilization and/or storage(especially when exposed to stress). This stabilising effect may arisefor a variety of reasons, though typically such stabilisers may act asosmolytes which mitigate against protein denaturation. Typicalstabilisers include amino acids (i.e. free amino acids not part of apeptide or protein—e.g. glycine, arginine, histidine, aspartic acid,lysine) and sugar stabilisers, such as a sugar polyol (e.g. mannitol,sorbitol), and/or a disaccharide (e.g. trehalose, sucrose, maltose,lactose), though the liquid pharmaceutical compositions of the inventioninclude a stabiliser, at least one of which is a sugar stabiliser (i.e.either a sugar polyol or a disaccharide). Most suitably the at least onesugar stabiliser is a non-reducing sugar (be it a sugar polyol or adisaccharide).

Herein, a “non-reducing sugar” is generally a sugar without any aldehydemoieties or without the capability of forming an aldehyde moiety (e.g.through isomerism).

Herein, a “tonicity modifier” or “tonicifier” refers to a reagent whoseinclusion within a composition suitably contributes to (or increases)the overall osmolality and osmolarity of the composition. Suitably, atonicifier, as used herein includes an agent which functions to render asolution similar in osmotic characteristics to physiologic fluids.

Herein, references to specific amounts of a given component of acomposition, especially a buffering agent, stabiliser, amino acid,surfactant, or tonicifier, suitably relate to the amounts of the pureanhydrous form of the relevant component (or compositions formed byusing said amounts of the pure anhydrous form), even though such acomponent may be used in a non-anhydrous form when forming thecomposition. Amounts of any corresponding non-anhydrous forms (e.g.monohydrates, dihydrates, etc.) may be readily calculated by simplyusing the appropriate multiplier. For instance, unless stated otherwise(as per the Examples, where quantities relate to trehalose dihydrate),amounts stipulated in relation to trehalose refer to the anhydrous formof trehalose (or compositions formed by using the stipulatedamounts/concentrations of anhydrous trehalose), which has a molecularweight of 342.296 g/mol, so to calculate the corresponding amount oftrehalose dihydrate needed to form the same composition (less waterwould have to be added) it is necessary to multiply the stipulatedamount by 378.33/342.296, since 378.33 is the molecular weight oftrehalose dihydrate. The skilled person would readily understand how tojudiciously adjust the quantity of diluent/water depending on the formof the components used, in order to derive the target concentrations.

Herein, the term “pharmaceutical composition” refers to a formulation ofa pharmaceutical active which renders the biological activity of theactive ingredient therapeutically effective, but which does not includeother ingredients which are obviously toxic to a subject to which theformulation are intended to be administered.

Herein, the term “stable” generally refers to the physical stabilityand/or chemical stability and/or biological stability of a component,typically an active or composition thereof, during preservation/storage.

It is to be appreciated that references to “treating” or “treatment”include prophylaxis as well as the alleviation of established symptomsof a condition. “Treating” or “treatment” of a state, disorder orcondition therefore includes: (1) preventing or delaying the appearanceof clinical symptoms of the state, disorder or condition developing in ahuman that may be afflicted with or predisposed to the state, disorderor condition but does not yet experience or display clinical orsubclinical symptoms of the state, disorder or condition, (2) inhibitingthe state, disorder or condition, i.e., arresting, reducing or delayingthe development of the disease or a relapse thereof (in case ofmaintenance treatment) or at least one clinical or subclinical symptomthereof, or (3) relieving or attenuating the disease, i.e., causingregression of the state, disorder or condition or at least one of itsclinical or subclinical symptoms.

In the context of the present invention, a “therapeutically effectiveamount” or “effective amount” of the antibody means an amount that iseffective, when administered to a mammal for treating a disease ordisorder, in prophylactic and therapeutic aspect and the antibody iseffective in treatment of the diseases concerned.

The “therapeutically effective amount” will vary depending on thecompound, the disease and its severity and the age, weight, etc., of themammal to be treated.

The term “human TNF-α” refers to the human cytokine which exists in a 17kD secreted form and a 26 kD membrane-associated form, and in abiologically active form, TNF-α could be observed as a trimer ofcovalently-bound 17 kD molecule. Its specific structure can be found inPennica, D. et al. (1984) Nature 312: 724-729; Davis, J. M. et al.(1987) Biochemistry 26, 1322-1326; and Jones, E. Y. et al. (1989) Nature338: 225-228.

The term “recombinant human antibody” is intended to include a humanantibody prepared, expressed, produced or isolated using a recombinantmethod.

Herein, amounts stipulated for components and ingredients, whetherspecified in terms of “parts”, ppm (parts per million), percentages (%,e.g. wt %), or ratios, are intended to be by weight, unless statedotherwise.

Where the quantity or concentration of a particular component of a givencomposition is specified as a weight percentage (wt % or % w/w), saidweight percentage refers to the percentage of said component by weightrelative to the total weight of the composition as a whole. It will beunderstood by those skilled in the art that the sum of weightpercentages of all components of a composition (whether or notspecified) will total 100 wt %. However, where not all components arelisted (e.g. where compositions are said to “comprise” one or moreparticular components), the weight percentage balance may optionally bemade up to 100 wt % by unspecified ingredients (e.g. a diluent, such aswater, or other non-essentially but suitable additives).

Herein, unless stated otherwise, the term “parts” (e.g. parts by weight,pbw) when used in relation to multiple ingredients/components, refers torelative ratios between said multiple ingredients/components. Expressingmolar or weight ratios of two, three or more components gives rise tothe same effect (e.g. a molar ratio of x, y, and z is x₁:y₁:z₁respectively, or a range x₁-x₂:y₁-y₂:z₁-z₂). Though in many embodimentsthe amounts of individual components within a composition may be givenas a “wt %” value, in alternative embodiments any or all such wt %values may be converted to parts by weight (or relative ratios) todefine a multi-component composition. This is so because the relativeratios between components is often more important than the absoluteconcentrations thereof in the liquid pharmaceutical compositions of theinvention. Where a composition comprising multiple ingredients isdescribed in terms of parts by weight alone (i.e. to indicate onlyrelative ratios of ingredients), it is not necessary to stipulate theabsolute amounts or concentrations of said ingredients (whether in totoor individually) because the advantages of the invention can stem fromthe relative ratios of the respective ingredients rather than theirabsolute quantities or concentrations. However, in certain embodiments,such compositions consists essentially of or consist of the stipulatedingredients and a diluents (e.g. water).

Where a composition is said to comprise a plurality of stipulatedingredients (optionally in stipulated amounts of concentrations), saidcomposition may optionally include additional ingredients other thanthose stipulated. However, in certain embodiments, a composition said tocomprise a plurality of stipulated ingredients may in fact consistessentially of or consist of all the stipulated ingredients.

Herein, where a composition is said to “consists essentially of” aparticular component, said composition suitably comprises at least 70 wt% of said component, suitably at least 90 wt % thereof, suitably atleast 95 wt % thereof, most suitably at least 99 wt % thereof. Suitably,a composition said to “consist essentially of” a particular componentconsists of said component save for one or more trace impurities.

Herein, the term “particle size” or “pore size” refers respectively tothe length of the longest dimension of a given particle or pore. Bothsizes may be measured using a laser particle size analyser and/orelectron microscopes (e.g. tunneling electron microscope, TEM, orscanning electron microscope, SEM). The particle count (for any givensize) can be obtained using the protocols and equipment outlined in theExamples, which relates to the particle count of sub-visible particles.

Liquid Pharmaceutical Composition

The present invention provides a liquid pharmaceutical composition,suitably as defined herein. The composition suitably comprises a humanmonoclonal antibody, suitably one which inhibits human TNF-α activity,suitably so as to prevent it from activating TNF receptors. Mostsuitably the liquid pharmaceutical composition comprises adalimumab,which in itself suitably includes any biosimilar thereof. Thecomposition suitably comprises an histidine buffering agent (orhistidine buffer system). The composition suitably comprises a sugarstabiliser. The composition suitably has a pH greater than or equal topH 6.30. The composition is suitably (substantially or entirely) free ofarginine or comprises arginine either in a concentration of at most 0.1mM, in a molar ratio of arginine to histidine buffering agent (orhistidine buffer system) of at most 1:150, or in a weight ratio ofarginine to adalimumab of at most 1:3000 (i.e. less than or equal to onepart by weight of histidine for every 3000 parts by weight histidinebuffering agent). Alternatively or in addition, the composition maysuitably include any one or more additional components defined herein inrelation to a liquid pharmaceutical composition (e.g. includingtonicifier, excluding arginine, etc.), optionally in any amount,concentration, or form stipulated herein; and wherein the compositionoptionally exhibits any one or more parameters or properties givenherein in relation to a liquid pharmaceutical composition (e.g. pH,osmolality).

Advantageously, the present invention provides alternative and improvedliquid pharmaceutical compositions, which generally exhibit betterstability and viability than those of the prior art. As is illustratedherein (see Examples), the liquid pharmaceutical formulations of thepresent invention have comparable or improved characteristics whencompared to the conventional formulations of adalimumab, for example thecommercially available formulation Humira®, when subjected to differentstressing conditions (thermal, mechanical and light). Their performanceis also generally comparable or better than many other comparativeformulations that were subjected to the same stress testing. Since thesestressing conditions are highly representative of the kind of stresssuch formulations are subjected to during manufacture, transport, andstorage, they provide an excellent indication of the advantages of theinvention. That such good stability performance can be achieved usingless complex formulations with fewer excipients was consideredsurprising in view of the general teachings of the prior art.

Adalimumab

Adalimumab, which is commercially available in HUMIRA® formulations, andits method of manufacture, is described in WO97/29131 (BASF) as D2E7,and elsewhere in the art. It is described as having “a light chain CDR3domain comprising the amino acid sequence of SEQ ID NO: 3 and a heavychain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4”(WO97/29131). Furthermore, the D2E7 antibody is described as having alight chain variable region (LCVR) comprising the amino acid sequence ofSEQ ID NO: 1 and a heavy chain variable region (HCVR) comprising theamino acid sequence of SEQ ID NO: 2 (WO97/29131).

The medical indications and function of Adalimumab, are elucidatedhereinbefore.

In the context of the invention “adalimumab” includes biosimilars, asdefined herein before, and the skilled person would readily appreciatethe scope of the term “adalimumab” in the context of the invention.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab at a concentration of from about 5 to about 150 mg/ml,suitably from about 25 to about 75 mg/mL. For example, the adalimumabmay be present in the formulation at a concentration of about 25, about30, about 35, about 40, about 45, about 50, about 55, about 60, about65, about 70 or about 75 mg/ml. In an embodiment, the adalimumab ispresent at a concentration from about 45 to about 55 mg/ml. In anembodiment, the adalimumab is present at a concentration of about 50mg/ml.

Buffer, Buffering Agent, and pH

Suitably, the liquid pharmaceutical composition is a buffered solutionwhose pH is stabilised by a buffering agent (or buffer system), suitablyin combination with an acid/base conjugate of the buffering agent. Assuch, the liquid pharmaceutical composition suitably comprises abuffering agent as defined herein. Preferably, the liquid pharmaceuticalcomposition additionally comprises an acid/base conjugate, wherein saidacid/base conjugate corresponds to the conjugate acid or conjugate baseof the buffering agent, depending on whether the buffering agent isitself a base or acid respectively. Collectively, the buffering agentand its acid/base conjugate may be considered a “buffer system”. Theliquid pharmaceutical composition thus suitably comprises a “buffersystem” (suitably comprising a buffering agent(s) and an acid/baseconjugate(s) thereof), and any concentrations stipulated in relation tothe buffer system generally relate to the combined concentrations of thebuffering agent(s) and any acid/base conjugate(s) thereof. Any “buffersystem” suitably comprises a weak acid and a weak base (see abovedefinitions).

Suitably, the buffering agent is an histidine buffering agent. Suitablythe histidine buffering agent is histidine (or a salt thereof), mostsuitably free histidine (e.g. zwitterionic histidine).

Suitably, the liquid pharmaceutical composition comprises an acid/baseconjugate of the buffering agent. This is less straightforward forhistidine buffering agents than many other common carboxylicacid/carboxylate buffer systems, since the imidazole moiety of histidinemeans that histidine generally exists in aqueous solution as anequilibrium mixture of protonated (imidazolium) and deprotonated (freeimidazole) forms a pHs between pH6-7. The protonated (imidazolium) formof histidine may be associated with one or more pharmaceuticallyacceptable anions—including anions such as hydroxide or chloride—thoughthe imidazolium form may additionally or alternatively exist in adiluents (e.g. water) as a solvated cation. As such, the protonated(imidazolium) form of histidine may be considered to be histidine'sacid/base conjugate, since it represents the conjugate acid ofhistidine. This conjugate acid of histidine suitably has both the aminoand imidazole group protonated but carboxylate group deprotonated—thisgives a net positively charge of +1). The combination of the bufferingagent and its acid/base conjugate constitutes a buffer system. Suitably,the liquid pharmaceutical composition comprises the buffering agent andits corresponding acid/base conjugate, suitably such that together thebuffering agent and its acid/base conjugate are present at a level (i.e.absolute amount or concentration) and in a relative amount (orconcentration) sufficient to provide the desired pH for the composition.The buffer system may be formed by simply mixing the buffering agent(e.g. histidine) with its acid/base conjugate (e.g. imidazolium saltform of histidine, e.g. histidine monohydrochloride), suitably inappropriate quantities to furnish a composition with the desired pH.Alternatively, the buffer system may be formed by mixing an acid or basewith either the buffering agent or its acid/base conjugate in order toform in situ the desired mixture of buffering agent and acid/baseconjugate. For example, the buffer system may be formed by adding a base(e.g. sodium hydroxide) to the buffering agent (e.g. histidine, whichmay self-equilibrate immediately when dissolved in water to yield bothhistidine and its conjugate acid), suitably in an amount appropriate tofurnish the desired pH and mixture of the buffering agent (e.g.histidine) and corresponding acid/base conjugate (i.e. imidazolium saltform of histidine). Alternatively, either method of forming the buffersystem may be employed, and pH may be judiciously adjusted by eitheradding further acid (suitably strong acid, such as HCl) or further base(suitably strong base, such as sodium hydroxide) until the required pHis reached.

As disclosed above, a “pH adjuster” may be used in conjunction withhistidine (or an imidazolium histidine salt, e.g. histidinehydrochloride) to obtain a desired pH. The pH adjuster may be a strongacid or a strong base, though it is preferably a strong base, such assodium hydroxide.

Most suitably, the buffer system is an histidine buffer system, suitablycomprising histidine in equilibrium with its imidazolium form.

Suitably, the liquid pharmaceutical composition comprises at most onebuffering agent. Suitably, the liquid pharmaceutical compositioncomprises at most one buffer system.

Suitably, the liquid pharmaceutical composition has a pH greater than orequal to 5.0. Suitably, the liquid pharmaceutical composition has a pHgreater than or equal to 6.3. Suitably, the liquid pharmaceuticalcomposition has a pH less than or equal to 6.7.

In a particular embodiment, especially where the buffering agent is anhistidine buffering agent, the liquid pharmaceutical composition has apH between 6.0 and 6.6. In a particular embodiment, the liquidpharmaceutical composition has a pH between 6.3 and 6.5. In a particularembodiment, the liquid pharmaceutical composition has a pH of about 6.4.

Suitably, the liquid pharmaceutical composition comprises a buffersystem (suitably a histidine buffer system comprising a histidinebuffering agent) at a concentration of from about 2 to about 50 mM. Inan embodiment, the buffer system is present at a concentration ofbetween 5 and 14 mM, most suitably about 10 mM. In an embodiment, thebuffer system/buffering agent(s) is present at a concentration of 10 mM.In an embodiment, the liquid pharmaceutical composition compriseshistidine (and/or a salt thereof) at a concentration of 10 mM. Thissuitably includes where the “buffering agent(s)” (e.g. histidine) isformed by the addition of a strong base (e.g. sodium hydroxide) to theconjugate acid of the buffering agent(s) (e.g. imidazolium form ofhistidine).

Suitably, the liquid pharmaceutical composition comprises the bufferingspecies (suitably histidine buffering species—e.g. histidine itself) ata concentration of from about 0.31 mg/mL to about 7.8 mg/mL. In anembodiment, the buffering species is present at a concentration ofbetween 0.77 mg/mL and 2.2 mg/mL, most suitably about 1.55 mg/mL. In anembodiment, the buffer system/buffering agent is present at aconcentration of 1.55 mg/mL. This includes where the “buffering agent”(e.g. histidine) is formed by the addition of a strong base (e.g. sodiumhydroxide) to the conjugate acid of the buffering agent (e.g.imidazolium form of histidine).

Suitably, the liquid pharmaceutical composition comprises the buffersystem (suitably the histidine buffer system) in a molar ratio of buffersystem to adalimumab of from about 5:1 to about 145:1. In an embodiment,the buffer system is present in a molar ratio of buffer system toadalimumab of from about 14:1 to about 40:1, most suitably about 29:1.In an embodiment, the buffer system/buffering agent(s) is present at aconcentration of 29:1. This includes where the “buffering agent(s)”(e.g. histidine) is formed by the addition of a strong base (e.g. sodiumhydroxide) to the conjugate acid of the buffering agent (e.g.imidazolium form of histidine—e.g. histidine monohydrochloride).

As illustrated in the Example section, liquid pharmaceuticalcompositions of the invention including an histidine bufferingagent/buffer system perform particularly well in stress tests,especially in relation to fragmentation and protein unfolding, which canbe important indicators of stability and drug product viability.Furthermore, liquid pharmaceutical compositions whose histidine buffersystem maintains a steady pH 6.4 perform particularly well.

Sugar Stabiliser

Suitably, the liquid pharmaceutical composition comprises a stabiliser,most suitably a sugar stabiliser. Suitably, such a component facilitatesmaintainance of the structural integrity of the biopharmaceutical drug,particularly during freezing and/or lyophilization and/or storage(especially when exposed to stress).

The liquid pharmaceutical composition may comprise one or more sugarstabilisers, though in preferred embodiments only a single sugarstabiliser is present.

Suitably, the sugar stabiliser is a sugar polyol (including sugaralcohols) and/or a disaccharide.

The sugar stabiliser is suitably selected from the group includingtrehalose, mannitol, sucrose, sorbitol, maltose, lactose, xylitol,arabitol, erythritol, lactitol, maltitol, inositol.

In a particular embodiment, the sugar stabiliser is selected from thegroup including trehalose, mannitol, sucrose, maltose, lactose, xylitol,arabitol, erythritol, lactitol, maltitol, inositol.

In a particular embodiment, the sugar stabiliser is a non-reducingsugar, optionally a non-reducing sugar listed anywhere herein.

In a particular embodiment, the sugar stabiliser is selected from thegroup including trehalose and mannitol.

In a particular embodiment, the sugar stabiliser is trehalose. Trehaloseis a particularly advantageous sugar stabiliser for use alongside anhistidine buffering agent/buffer system in liquid adalimumabformulations.

Suitably, the liquid pharmaceutical composition comprises at most onesugar stabiliser, suitably at most one sugar polyol and/or disaccharide.Suitably, the liquid pharmaceutical composition comprises trehalose asthe only sugar stabiliser.

Suitably the trehalose used to form the liquid pharmaceuticalcomposition is trehalose dihydrate, though suitably any amountsstipulated in relation to trehalose (unless stated otherwise—as done inthe Examples) pertain to pure, anhydrous trehalose. Such amounts may beconverted into an amount of trehalose dihydrate by applying anappropriate multiplier. Moreover, for the purposes of assessing whethera given formulation falls within the scope of any of the trehalosequantity definitions given herein, an amount of trehalose dihydrate canbe readily converted into a corresponding amount of pure, anhydroustrehalose (with an equal number of moles) through applying saidmultiplier in reverse. This principle may be adopted for any sugarstabiliser component. Concentrations, when given as a molarconcentration, will of course be the same regardless of the hydrationstate of the sugar stabiliser.

Suitably, the liquid pharmaceutical composition comprises the sugarstabilizer(s) (most suitably trehalose) at a concentration of from about50 to about 400 mM, more suitably from about 100 to about 300 mM, moresuitably from about 150 to about 250 mM. In an embodiment, the sugarstabilizer(s) is present at a concentration of between 190 and 210 mM,most suitably about 200 mM. In an embodiment, trehalose is present at aconcentration of 200 mM.

Suitably, the liquid pharmaceutical composition comprises the sugarstabilizer(s) (most suitably trehalose) at a concentration of from about15 mg/mL to about 140 mg/mL, more suitably from about 35 mg/mL to about100 mg/mL, more suitably from about 45 mg/mL to about 80 mg/mL. In anembodiment, the sugar stabilizer(s) is present at a concentration ofbetween 65 mg/mL and 72 mg/mL, most suitably about 68 mg/mL. In aparticular embodiment, trehalose is present at a concentration of about68 mg/mL (which equates to about 75.7 mg/mL trehalose dihydrate).

Suitably, the liquid pharmaceutical composition comprises the sugarstabilizer(s) (most suitably trehalose) in a molar ratio of sugarstabilizer(s) to adalimumab of from about 145:1 to about 1150:1, moresuitably from about 290:1 to about 860:1, more suitably from about 430:1to about 720:1. In an embodiment, the sugar stabilizer(s) is present ata molar ratio of sugar stabilizer(s) to adalimumab of from about 550:1to about 605:1, most suitably about 576:1. In an embodiment, trehaloseis present at a molar ratio of trehalose to adalimumab of about 576:1.

As illustrated in the Example section, liquid pharmaceuticalcompositions of the invention including a sugar stabiliser as definedherein perform particularly well in stress tests, especially in relationto aggregation, fragmentation and protein unfolding, which can beimportant indicators of stability and drug product viability.Furthermore, liquid pharmaceutical compositions comprising trehalose asthe sugar stabiliser perform particularly well.

Diluent

The liquid pharmaceutical compositions of the invention may include anyone or more pharmaceutically acceptable diluents, or mixture thereof.However, most suitably the liquid pharmaceutical composition is anaqueous pharmaceutical composition. Most suitably the diluent is water,and suitably water alone. The water is suitably water for injection(WFI).

Suitably the diluent may constitute the balance of ingredients in anyliquid pharmaceutical composition, for instance so that the weightpercentages total 100%. Suitably any concentrations given herein inrelation to any component of the liquid pharmaceutical compositionrepresent concentrations of said component in (and suitably dissolvedin) the diluent in admixture with any other components.

The liquid pharmaceutical composition of the invention is suitably asolution, and is suitably (substantially or entirely) free ofparticulates or precipitates.

Absent or Low Level Components Low/No Arginine

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of arginine (suitably L-arginine) or comprisesarginine in a concentration of at most 0.1 mM, more suitably at most0.01 mM, most suitably at most 0.001 mM.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of arginine or comprises arginine in a molar ratio ofarginine to buffering agent (or buffer system) of at most 1:150 (i.e.less than or equal to one mole of arginine for every 150 moles ofbuffering agent or buffer system), more suitably at most 1:1500, mostsuitably at most 1:15,000.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of arginine or comprises arginine in a weight ratio ofarginine to adalimumab of at most 1:3000 (i.e. less than or equal to onepart by weight of arginine for every 3000 parts by weight adalimumab),more suitably at most 1:30,000, most suitably at most 1:300,000.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of arginine or comprises arginine in a molar ratio ofarginine to adalimumab of at most 1:3.75 (i.e. less than or equal to onemole of arginine for every 3.75 moles adalimumab), more suitably at most1:37.5, most suitably at most 1:375.

As explained herein, such references to “arginine” in the context oftheir presence or otherwise within liquid pharmaceutical compositionsrelate to the corresponding free amino acid(s) and not amino acidresidue(s) covalently incorporated as part of a larger compound, such asa peptide or protein.

As illustrated in the Example section, liquid pharmaceuticalcompositions of the invention which (substantially or entirely) excludearginine perform particularly well in stress tests, especially inrelation to aggregation, fragmentation and protein unfolding.

Low/No Amino Acids

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of amino acids other than histidine (which is suitablythe buffering agent) or comprises one or more amino acids other thanhistidine in a (collective) concentration of at most 0.1 mM, moresuitably at most 0.01 mM, most suitably at most 0.001 mM.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of amino acids other than histidine or comprises oneor more amino acids other than histidine in a (collective) molar ratioof amino acids(s) to buffering agent (or buffer system) of at most 1:150(i.e. less than or equal to one mole of amino acids(s) other thanhistidine for every 150 moles of buffering agent or buffer system), moresuitably at most 1:1500, most suitably at most 1:15,000.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of amino acids other than histidine or comprises oneor more amino acids other than histidine in a (collective) weight ratioof amino acids(s) to adalimumab of at most 1:3000 (i.e. less than orequal to one part by weight of amino acids(s) other than histidine forevery 3000 parts by weight adalimumab), more suitably at most 1:30,000,most suitably at most 1:300,000.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of amino acids other than histidine or comprises oneor more amino acids other than histidine in a (collective) molar ratioof amino acid(s) to adalimumab of at most 1:3.75 (i.e. less than orequal to one mole of amino acid(s) other than histidine for every 3.75moles adalimumab), more suitably at most 1:37.5, most suitably at most1:375.

As explained herein, such references to “amino acids” in the context oftheir presence or otherwise within liquid pharmaceutical compositionsrelate to the corresponding free amino acid(s) and not amino acidresidue(s) covalently incorporated as part of a larger compound, such asa peptide or protein.

Suitably, the amino acids referred to in this section (and deemed eitherabsent or present in low quantities) may be natural and/or artificialamino acids, though they are preferably natural amino acids. Inparticular, the liquid pharmaceutical compositions are either(substantially or entirely) free of any amino acids selected from thegroup including: arginine, lysine, and aspartic acid; or comprises oneor more of the aforesaid amino acids in an amount, concentration, molarratio, or weight ratio as hereinbefore defined in relation to “aminoacid(s) other than histidine”.

As illustrated in the Example section, liquid pharmaceuticalcompositions of the invention which (substantially or entirely) excludeamino acids other than histidine or certain amino acids, as definedabove, perform particularly well in stress tests, especially in relationto aggregation, fragmentation and protein unfolding.

Low/No Surfactants

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of surfactants (whether cationic, anionic, amphoteric,or non-ionic) with the optional exception of polysorbate 80(polyoxyethylene (20) sorbitan monooleate) or comprises one or more ofsaid surfactants (optionally excluding polysorbate 80) in a (collective)concentration of at most 1 mM, more suitably at most 0.1 mM, moresuitably at most 0.01 mM, more suitably at most 0.001 mM, most suitablyat most 0.0001 mM. The liquid pharmaceutical composition may, under suchcircumstances, optionally comprise polysorbate 80 as defined herein.However, in preferred embodiments, the liquid pharmaceutical compositionis (substantially or entirely) free of polysorbate 80 or comprisespolysorbate 80 only in the limited amounts/concentrations mentionedabove, suitably collectively with any other surfactants.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of surfactants (whether cationic, anionic, amphoteric,or non-ionic) with the optional exception of polysorbate 80(polyoxyethylene (20) sorbitan monooleate) or comprises one or more ofsaid surfactants (optionally excluding polysorbate 80) in a (collective)molar ratio of surfactant(s) to buffering agent (or buffer system) of atmost 1:10, more suitably at most 1:100, most suitably at most 1:1000,more suitably at most 1:10,000, suitably at most 1:100,000. The liquidpharmaceutical composition may, under such circumstances, optionallycomprise polysorbate 80 as defined herein. However, in preferredembodiments, the liquid pharmaceutical composition is (substantially orentirely) free of polysorbate 80 or comprises polysorbate 80 only in thelimited amounts/concentrations mentioned above, suitably collectivelywith any other surfactants.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of surfactants (whether cationic, anionic, amphoteric,or non-ionic) with the optional exception of polysorbate 80(polyoxyethylene (20) sorbitan monooleate) or comprises one or more ofsaid surfactants (optionally excluding polysorbate 80) in a (collective)weight ratio of surfactant(s) to adalimumab of at most 1:50 (i.e. lessthan or equal to one part by weight of surfactant(s) for every 50 partsby weight adalimumab), more suitably at most 1:500, more suitably atmost 1:5000, more suitably at most 1:50,000, suitably at most 1:500,000.The liquid pharmaceutical composition may, under such circumstances,optionally comprise polysorbate 80 as defined herein. However, inpreferred embodiments, the liquid pharmaceutical composition is(substantially or entirely) free of polysorbate 80 or comprisespolysorbate 80 only in the limited amounts/concentrations mentionedabove, suitably collectively with any other surfactants.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of surfactants (whether cationic, anionic, amphoteric,or non-ionic) with the optional exception of polysorbate 80(polyoxyethylene (20) sorbitan monooleate) or comprises one or more ofsaid surfactants (optionally excluding polysorbate 80) in a (collective)molar ratio of surfactant(s) to adalimumab of at most 3:1, more suitablyat most 0.3:1, more suitably 0.003:1, more suitably 0.0003:1, suitably0.00003:1. The liquid pharmaceutical composition may, under suchcircumstances, optionally comprise polysorbate 80 as defined herein.However, in preferred embodiments, the liquid pharmaceutical compositionis (substantially or entirely) free of polysorbate 80 or comprisespolysorbate 80 only in the limited amounts/concentrations mentionedabove, suitably collectively with any other surfactants.

Suitably, the surfactants referred to in this section (and deemed eitherabsent or present in low quantities) may be cationic, anionic,amphoteric, or non-ionic surfactants. Suitably, the surfactants referredto in this section (and deemed either absent or present in lowquantities) include cationic, anionic, and amphoteric surfactants, butmay optionally exclude non-ionic surfactants (e.g. polysorbates orspans) or at least may optionally exclude polysorbate 80. As such, theliquid pharmaceutical composition is either (substantially or entirely)free of cationic, anionic, or amphoteric surfactants or comprises one ormore of said surfactants in an amount, concentration, molar ratio, orweight ratio of at most that stipulated in any of the precedingparagraphs of this sub-section in relation to “surfactant(s)” moregenerally.

The liquid pharmaceutical composition is either (substantially orentirely) free of non-ionic surfactants with the optional exception ofpolysorbate 80 or comprises one or more of said surfactants in anamount, concentration, molar ratio, or weight ratio of at most thatstipulated in any of the preceding paragraphs of this sub-section inrelation to “surfactant(s)” more generally.

The liquid pharmaceutical composition is either (substantially orentirely) free of polysorbate surfactants with the optional exception ofpolysorbate 80 or comprises one or more of said surfactants in anamount, concentration, molar ratio, or weight ratio of at most thatstipulated in any of the preceding paragraphs of this sub-section inrelation to “surfactant(s)” more generally. The liquid pharmaceuticalcomposition may, under such circumstances, optionally comprisepolysorbate 80 as defined herein. However, in preferred embodiments, theliquid pharmaceutical composition is (substantially or entirely) free ofpolysorbate 80 or comprises polysorbate 80 only in the limitedamounts/concentrations mentioned above, suitably collectively with anyother surfactants.

The liquid pharmaceutical composition is either (substantially orentirely) free of polysorbate 20 (also known as Tween 20-polyoxyethylene(20) sorbitan monolaurate) surfactants or comprises one or more of saidsurfactants in an amount, concentration, molar ratio, or weight ratio ofat most that stipulated in any of the preceding paragraphs of thissub-section in relation to “surfactant(s)” more generally.

The liquid pharmaceutical composition may suitably be either(substantially or entirely) free of polysorbate 80 surfactants orcomprises said surfactant(s) in an amount, concentration, molar ratio,or weight ratio as hereinbefore defined in relation to “surfactant(s)”.The liquid pharmaceutical composition is either (substantially orentirely) free of polysorbate 80 surfactants or comprises one or more ofsaid surfactants in an amount, concentration, molar ratio, or weightratio of at most that stipulated in any of the preceding paragraphs ofthis sub-section in relation to “surfactant(s)” more generally.

As illustrated in the Example section, liquid pharmaceuticalcompositions of the invention which (substantially or entirely) excludesurfactants or certain surfactants, as defined above, performparticularly well in stress tests, especially in relation toaggregation, fragmentation and protein unfolding.

Low/No Phosphate

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of phosphate buffering agents (e.g. sodium dihydrogenphosphate, disodium hydrogen phosphate) or comprises a phosphate buffersystem in a concentration of at most 0.1 mM, more suitably at most 0.01mM, most suitably at most 0.001 mM.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of phosphate buffering agents (e.g. sodium dihydrogenphosphate, disodium hydrogen phosphate) or comprises a phosphate buffersystem in a molar ratio of phosphate buffer system to any non-phosphatebuffer systems present of at most 1:150 (i.e. less than or equal to onemole of phosphate buffer system for every 150 moles of non-phosphatebuffer system present), more suitably at most 1:1500, most suitably atmost 1:15,000.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of phosphate buffering agents or comprises a phosphatebuffer system in a molar ratio of phosphate buffer system to adalimumabof at most 1:3.75 (i.e. less than or equal to one mole of phosphatebuffer system for every 3.75 moles adalimumab), more suitably at most1:37.5, most suitably at most 1:375.

References to “phosphate buffering agents” in the context of theirpresence or otherwise within liquid pharmaceutical compositions relateto any phosphate salts in any form or protonation state, includingphosphate, monohydrogen phosphate, and dihydrogen phosphate. It does,however, suitably exclude any phosphate moieties or residues that may becovalently incorporated as part of a larger compound, such as aphosphorylated or glycosylated peptide or protein.

As illustrated in the Example section, liquid pharmaceuticalcompositions of the invention which (substantially or entirely) excludephosphate buffering agents perform particularly well in stress tests,especially in relation to aggregation, fragmentation and proteinunfolding.

Optional Additional Components Tonicifier

The liquid pharmaceutical composition of the invention suitablycomprises a “tonicity modifier” (or “tonicifier”) or one or moretonicifiers, suitably as defined herein.

The inclusion of a tonicifier suitably contributes to (or increases) theoverall osmolality and osmolarity of the composition. Suitably atonicifier is present within the composition in a quantity orconcentration sufficient for the composition to be (substantially)isotonic with body fluids. Suitably a tonicifier is present within thecomposition in a quantity or concentration sufficient for thecomposition to have an osmolarity or osmolality within a range definedherein.

Any suitable tonicifier may be used. However, suitably the tonicifier isselected from the group including water-soluble metal salts (e.g. sodiumchloride, potassium chloride, magnesium chloride, calcium chloride),water-soluble tonicifying sugars/sugar alcohols (e.g. glucose, sucrose,mannitol), and/or other water-soluble polyols. Suitably thetonicifier(s) is non-buffering (i.e. gives rise to little or nobuffering effect). As such, any metal salt tonicifiers are suitably notbuffering agents.

The liquid pharmaceutical composition may comprise one or moretonicifiers, though preferably only a single “tonicifier” as such ispresent (notwithstanding any tonicifying effects imparted to thecomposition by components intended to serve another function as definedherein).

Most preferably, the tonicifier is or comprises a metal salt (preferablya non-buffering water-soluble metal salt). Suitably, said metal salt isor comprises a metal halide, suitably an alkali or an alkaline earthmetal halide, suitably an alkali metal chloride.

In a particular embodiment, the tonicifier is or comprises sodiumchloride. In a particular embodiment, the tonicifier is sodium chloride.Sodium chloride is a particularly advantageous stabiliser for usealongside an histidine buffering agent/buffer system in liquidadalimumab formulations.

Suitably, the liquid pharmaceutical composition comprises thetonicifier(s) (most suitably sodium chloride) at a concentration of fromabout 10 to about 200 mM, more suitably from about 20 to about 100 mM,more suitably from about 25 to about 75 mM. In an embodiment, thetonicifier(s) is present at a concentration of between 40 and 60 mM,most suitably about 50 mM. In an embodiment, sodium chloride is presentat a concentration of 50 mM.

Suitably, the liquid pharmaceutical composition comprises thetonicifier(s) (most suitably sodium chloride) at a concentration of fromabout 0.5 mg/mL to about 12 mg/mL, more suitably from about 1.2 mg/mL toabout 5 mg/mL, more suitably from about 1.5 mg/mL to about 4.4 mg/mL. Inan embodiment, the tonicifier(s) is present at a concentration ofbetween 2.7 mg/mL and 3.1 mg/mL, most suitably about 2.9 mg/mL. In aparticular embodiment, sodium chloride is present at a concentration ofabout 2.9 mg/mL.

Suitably, the liquid pharmaceutical composition comprises thetonicifier(s) (most suitably sodium chloride) in a molar ratio oftonicifier to adalimumab of from about 30:1 to about 580:1, moresuitably from about 60:1 to about 290:1, more suitably from about 70:1to about 220:1. In an embodiment, the tonicifier(s) is present at amolar ratio of tonicifier to adalimumab of from about 115:1 to about175:1, most suitably about 145:1. In an embodiment, sodium chloride ispresent at a molar ratio of sodium chloride to adalimumab of about145:1.

As illustrated in the Example section, liquid pharmaceuticalcompositions of the invention including a tonicifier as defined hereinperform particularly well in stress tests, especially in relation toaggregation, fragmentation and protein unfolding, which can be importantindicators of stability and drug product viability. Furthermore, liquidpharmaceutical compositions comprising sodium chloride, particularly inan amount range as stipulated, perform particularly well.

Surfactant

The liquid pharmaceutical composition of the invention may comprise asurfactant or one or more surfactants, suitably as defined herein.

Any suitable surfactant may be used. However, suitably the surfactant isa non-ionic surfactant, most suitably a polysorbate (polyoxyethyleneglycol sorbitan alkyl esters) or span (sorbitan alkyl esters)surfactant.

Though one or more surfactants may be included within the liquidpharmaceutical composition of the invention, most suitably only a singlesurfactant is present, most suitably a single non-ionic surfactant(suitably as defined herein).

The surfactant(s) are suitably selected from Polysorbate 20(Polyoxyethylene (20) sorbitan monolaurate), Polysorbate 40(Polyoxyethylene (20) sorbitan monopalmitate), Polysorbate 60(Polyoxyethylene (20) sorbitan monostearate), Polysorbate 80(Polyoxyethylene (20) sorbitan monooleate), Sorbitan monolaurate,Sorbitan monopalmitate, Sorbitan monostearate, Sorbitan tristearate,and/or Sorbitan monooleate.

In a particular embodiment, the surfactant(s) are selected fromPolysorbate 20, Polysorbate 40, Polysorbate 60, and/or Polysorbate 80.In a particular embodiment, the liquid pharmaceutical compositioncomprises a single surfactant selected from Polysorbate 20, Polysorbate40, Polysorbate 60, and Polysorbate 80.

In a particular embodiment, the surfactant is polysorbate 80 orpolysorbate 20. In a particular embodiment, the surfactant ispolysorbate 80.

Suitably, the liquid pharmaceutical composition comprises thesurfactant(s) (most suitably polysorbate 80) at a concentration of fromabout 0.0001 to about 5 mM (i.e. 0.1 μM-5 mM), more suitably from about0.001 to about 2 mM, more suitably from about 0.01 to about 1.0 mM. Inan embodiment, the surfactant(s) is present at a concentration ofbetween 0.72 and 0.80 mM, most suitably about 0.76 mM. In an embodiment,polysorbate 80 is present at a concentration of 0.76 mM.

Suitably, the liquid pharmaceutical composition comprises thesurfactant(s) (most suitably polysorbate 80) at a concentration of fromabout 0.001 mg/mL to about 5 mg/mL, more suitably from about 0.01 mg/mLto about 2 mg/mL, more suitably from about 0.05 mg/mL to about 1.5mg/mL. In an embodiment, the surfactant(s) is present at a concentrationof between 0.9 mg/mL and 1.1 mg/mL, most suitably about 1.0 mg/mL. In aparticular embodiment, polysorbate 80 is present at a concentration ofabout 1.0 mg/mL.

Suitably, the liquid pharmaceutical composition comprises thesurfactant(s) (most suitably polysorbate 80) in a molar ratio ofsurfactant(s) to adalimumab of from about 1:3500 to about 15:1, moresuitably from about 1:350 to about 6:1, more suitably from about 1:35 toabout 3:1. In an embodiment, the surfactant(s) is present at a molarratio of surfactant(s) to adalimumab of from about 2.1:1 to about 2.3:1,most suitably about 2.2:1. In an embodiment, polysorbate 80 is presentat a molar ratio of polysorbate 80 to adalimumab of about 2.2:1.

In preferred embodiments, however, the liquid pharmaceutical compositionis (substantially or entirely) free of polysorbate 80, and more suitably(substantially or entirely) free of any surfactants.

Other Parameters Relating to the Invention Osmolality

Suitably, the osmolality of the liquid pharmaceutical composition isbetween 200 and 400 mOsm/kg, more suitably between 220 and 390 mOsm/kg,more suitably between 230 and 350 mOsm/kg, more suitably between 240 and340 mOsm/kg, more suitably between 260 and 320 mOsm/kg, most suitablybetween 280 and 310 mOsm/kg. Suitably the relative amounts andconcentrations of the various components of the composition may bejudiciously tuned to achieve the desired osmolality, and the particularnovel combination of components allows this to be largely achievedwithout undermining other important parameters. However, suitably therelative amounts and concentrations of the various components of thecomposition may be selected so as to optimise other parameters—thepresent disclosure, including the examples and protocols set forththerein, enable the skilled person to achieve this end and to realise a,some, or all of the benefits of the present invention.

Protein Unfolding Temperature

Suitably, the protein unfolding temperature (suitably as measured viathe DSF protocols defined herein) of adalimumab in the liquidpharmaceutical composition of the invention is greater than or equal to65° C., more suitably greater than or equal to 70° C. The novelcombination of components present within the composition of theinvention enables the skilled person to achieve high unfoldingtemperatures, which may be considered desirable from a thermal stabilityperspective.

Parameters when Subjected to Thermal Stress

Suitably the quantity (or concentration) of aggregates (suitably derivedfrom adalimumab, and suitably as determined by the SE-HPLC protocols asdefined herein) present within the liquid pharmaceutical compositionincreases by no more than a factor of 4 (i.e. 4 times the amountrelative to an arbitrary start time) when the composition is thermallystressed at 40° C. (i.e. the composition is maintained at a temperatureof 40° C.) over a period of 28 days, suitably by no more than factor of3, suitably by no more than factor of 2.5, suitably by no more thanfactor of 2.2.

Suitably the quantity (or concentration) of fragments (suitably derivedfrom adalimumab and suitably measured via the bioanalyzer protocolsdefined herein) present within the liquid pharmaceutical compositionincreases by no more than a factor of 4 (i.e. 4 times the amountrelative to an arbitrary start time) when the composition is thermallystressed at 40° C. (i.e. the composition is maintained at a temperatureof 40° C.) over a period of 28 days, suitably by no more than factor of3, suitably by no more than factor of 2.5, suitably by no more thanfactor of 2.2.

Suitably the turbidity (suitably as measured via nephelometry inaccordance with the protocols set forth herein) of the liquidpharmaceutical composition increases by no more than a factor of 2 (i.e.2 times the amount relative to an arbitrary start time) when thecomposition is thermally stressed at 40° C. (i.e. the composition ismaintained at a temperature of 40° C.) over a period of 28 days,suitably by no more than a factor of 1.5, suitably by no more than afactor of 1.2, and suitably the turbity does not increase at all.

Suitably the pH of the liquid pharmaceutical composition changes(whether through increase or decrease, though generally by a decrease inpH) by no more than 0.5 pH units when the composition is thermallystressed at 40° C. (i.e. the composition is maintained at a temperatureof 40° C.) over a period of 28 days, suitably by no more than 0.2 pHunits, suitably by no more than 0.1 pH units, most suitably the pH doesnot change at all (to one decimal place).

Parameters when Subjected to Mechanical Stress

Suitably the quantity (or concentration) of aggregates (suitably derivedfrom adalimumab, and suitably as determined by the SE-HPLC protocols asdefined herein) present within the liquid pharmaceutical compositionincreases by no more than a factor of 2 (i.e. 2 times the amountrelative to an arbitrary start time) when the composition ismechanically stressed (i.e. shaken as per the protocols outlined herein)over a period of 48 hours, suitably by no more than factor of 1.5,suitably by no more than factor of 1.2, suitably by no more than factorof 1.1.

Suitably the quantity (or concentration) of fragments (suitably derivedfrom adalimumab and suitably measured via the bioanalyzer protocolsdefined herein) present within the liquid pharmaceutical compositionincreases by no more than a factor of 2 (i.e. 2 times the amountrelative to an arbitrary start time) when the composition ismechanically stressed (i.e. shaken as per the protocols outlined herein)over a period of 48 hours, suitably by no more than factor of 1.5,suitably by no more than factor of 1.2, suitably by no more than factorof 1.1.

Suitably the turbidity (suitably as measured via nephelometry inaccordance with the protocols set forth herein) of the liquidpharmaceutical composition increases by no more than a factor of 2 (i.e.2 times the amount relative to an arbitrary start time) when thecomposition is mechanically stressed (i.e. shaken as per the protocolsoutlined herein) over a period of 48 hours, suitably by no more thanfactor of 1.5, suitably by no more than factor of 1.2, suitably by nomore than factor of 1.1, and suitably the turbity does not increase atall.

Suitably the pH of the liquid pharmaceutical composition changes(whether through increase or decrease, though generally by a decrease inpH) by no more than 0.5 pH units when the composition is mechanicallystressed (i.e. shaken as per the protocols outlined herein) over aperiod of 48 hours, suitably by no more 0.2 pH units, suitably by nomore than 0.1 pH units, most suitably the pH does not change at all (toone decimal place).

Parameters when Subjected to Light Stress

Suitably the quantity (or concentration) of aggregates (suitably derivedfrom adalimumab, and suitably as determined by the SE-HPLC protocols asdefined herein) present within the liquid pharmaceutical compositionincreases by no more than a factor of 50 (i.e. 50 times the amountrelative to an arbitrary start time) when the composition is lightstressed (i.e. the composition is exposed to light in accordance withprotocols disclosed herein, i.e. 7 hours at 765 W/m²), suitably by nomore than factor of 45, suitably by no more than factor of 35, suitablyby no more than factor of 30.

Suitably the quantity (or concentration) of fragments (suitably derivedfrom adalimumab and suitably measured via the bioanalyzer protocolsdefined herein) present within the liquid pharmaceutical compositionincreases by no more than a factor of 4 (i.e. 4 times the amountrelative to an arbitrary start time) when the composition is lightstressed (i.e. the composition is exposed to light in accordance withprotocols disclosed herein, i.e. 7 hours at 765 W/m²), suitably by nomore than factor of 3, suitably by no more than factor of 2.5, suitablyby no more than factor of 2.

Suitably the turbidity (suitably as measured via nephelometry inaccordance with the protocols set forth herein) of the liquidpharmaceutical composition increases by no more than a factor of 2 (i.e.2 times the amount relative to an arbitrary start time) when thecomposition is light stressed (i.e. the composition is exposed to lightin accordance with protocols disclosed herein, i.e. 7 hours at 765W/m²), suitably by no more than a factor of 1.5, suitably by no morethan a factor of 1.2, and suitably the turbity does not increase at all.

Suitably the pH of the liquid pharmaceutical composition changes(whether through increase or decrease, though generally by a decrease inpH) by no more than 0.5 pH units when the composition is light stressed(i.e. the composition is exposed to light in accordance with protocolsdisclosed herein, i.e. 7 hours at 765 W/m²), suitably by no more than0.2 pH units, suitably by no more than 0.1 pH units, most suitably thepH does not change at all (to one decimal place).

Parameters when Subjected to Freeze/Thaw Cycles

Suitably the quantity (or concentration) of aggregates (suitably derivedfrom adalimumab, and suitably as determined by the SE-HPLC protocols asdefined herein) present within the liquid pharmaceutical compositionincreases by no more than a factor of 1.5 (i.e. 1.5 times the amountrelative to an arbitrary start time) when the composition is subjectedto five freeze/thaw cycles (i.e. the composition is frozen and thawedfive times in accordance with protocols disclosed herein, i.e. −80° C.to 20° C. five times), suitably by no more than factor of 1.2, suitablyby no more than factor of 1.1, suitably by there is (substantially) noincrease at all in the quantity (or concentration) of aggregates.

Suitably the quantity (or concentration) of sub-visible particles orprecipitates, with a particle size less than or equal to 25 microns,present within the liquid pharmaceutical composition increases by nomore than a factor of 4 (i.e. 4 times the amount relative to anarbitrary start time) when the composition is subjected to fivefreeze/thaw cycles (i.e. the composition is frozen and thawed five timesin accordance with protocols disclosed herein, i.e. −80° C. to 20° C.five times), suitably by no more than factor of 3, suitably by no morethan factor of 2.5, suitably by no more than factor of 2.2. Suitably thequantity (or concentration) of sub-visible particles or precipitates,with a particle size less than or equal to 10 microns, present withinthe liquid pharmaceutical composition increases by no more than a factorof 4 (i.e. 4 times the amount relative to an arbitrary start time) whenthe composition is subjected to five freeze/thaw cycles (i.e. thecomposition is frozen and thawed five times in accordance with protocolsdisclosed herein, i.e. −80° C. to 20° C. five times), suitably by nomore than factor of 3, suitably by no more than factor of 2.5, suitablyby no more than factor of 2.2.

Suitably the quantity (or concentration) of sub-visible particles orprecipitates, with a particle size less than or equal to 25 microns,present within the liquid pharmaceutical composition increases by nomore than a factor of 4 (i.e. 4 times the amount relative to anarbitrary start time) when the composition is subjected to 5 freeze/thawcycles, suitably by no more than factor of 3, suitably by no more thanfactor of 2.5, suitably by no more than factor of 2.2. Suitably thequantity (or concentration) of sub-visible particles or precipitates,with a particle size less than or equal to 10 microns, present withinthe liquid pharmaceutical composition increases by no more than a factorof 4 (i.e. 4 times the amount relative to an arbitrary start time) whenthe composition is subjected to 5 freeze/thaw cycles, suitably by nomore than factor of 3, suitably by no more than factor of 2.5, suitablyby no more than factor of 2.2.

Methods of Stabilising Antibody

In view of the aforementioned points in this sub-section, and the datapresented in the examples, the present invention also provides a methodof stabilising liquid adalimumab compositions (chemically and/orphysically optionally in relation to any one or more of theaforementioned parameters/properties), comprising mixing with adalimumabwith any relevant components required to form a liquid pharmaceuticalcomposition as defined herein. Different embodiments will suitablyrequire different combinations of components to be mixed, potentially indifferent amounts, and the skilled person can readily deduce suchcombinations and amounts by reference to the foregoing disclosurerelating to the liquid pharmaceutical composition. Such differentcombinations of components may stabilise liquid adalimumab compositionsin different respects. For instance, mixing with adalimumab with theaforementioned components to form a liquid pharmaceutical composition asdefined herein may stabilise adalimumab by:

-   -   i) Increasing the protein unfolding temperature of adalimumab;    -   ii) Inhibiting the formation of aggregates;    -   iii) Inhibiting the formation of fragments;    -   iv) Inhibiting the formation of sub-visible particles (either        525 microns or 510 microns);    -   v) Inhibiting turbidification;    -   vi) Inhibiting pH changes;    -   vii) Inhibiting photo-oxidation; and/or    -   viii) Reducing instability upon freeze/thaw cycles.

As such, the present invention provides a method of achieving one, some,or all of the following benefits:

-   -   i) Increased protein unfolding temperatures for adalimumab;    -   ii) Inhibition of formation of aggregates;    -   iii) Inhibition of formation of fragments;    -   iv) Inhibition of formation of sub-visible particles (either 525        microns or 510 microns);    -   v) Inhibition of turbidification;    -   vi) Inhibition of pH changes;    -   vii) Inhibition of photo-oxidation; and/or    -   viii) Reduced instability upon freeze/thaw cycles;        the method comprising manufacturing a liquid pharmaceutical        composition of adalimumab as defined herein.

Suitably, the liquid pharmaceutical compositions of the invention have ashelf life of at least 6 months, suitably at least 12 months, suitablyat least 18 months, more suitably at least 24 months. Suitably, theliquid pharmaceutical compositions of the invention have a shelf life ofat least 6 months, suitably at least 12 months, suitably at least 18months, more suitably at least 24 months, at a temperature of 2-8° C.

Enabling the Skilled Person to Optimise Key Stability Properties

The novel combination of components disclosed for use in liquidpharmaceutical compositions of the invention enables the skilled personto produce (and judiciously fine-tube) compositions which exhibitcomparable or enhanced properties relative to compositions of the priorart. In particular, the present disclosure now provides the skilledperson with all the necessary tools to optimise formulation stability,and in particular optimise one or more of: inhibition of aggregation,fragmentation, protein unfolding, precipitation, pH slippage, andoxidation (especially photo-oxidation). Furthermore, the skilled personis given guidance on how to achieve such optimisations (throughjudiciously varying the compositions) and how, in the process, tominimise any detrimental side-effects. The present disclosure enablesthe skilled person to work across the scope of the invention to producea variety of specific compositions which exhibit comparable or improvedproperties relative to compositions of the prior art, and this can beachieved using fewer components.

Particular Embodiments

In an embodiment, the liquid pharmaceutical composition comprises:

-   -   adalimumab;    -   an histidine buffering agent (e.g. histidine) (or histidine        buffer system);    -   a sugar stabiliser (e.g. trehalose); and    -   a surfactant (e.g. polysorbate 80).

In an embodiment, the liquid pharmaceutical composition comprises:

-   -   adalimumab;    -   an histidine buffering agent (e.g. histidine) (or histidine        buffer system);    -   a sugar stabiliser (e.g. trehalose);    -   a tonicifier (e.g. sodium chloride); and    -   optionally a surfactant (e.g. polysorbate 80).

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, histidine buffering agent (or buffer system), and a sugarstabiliser in a molar ratio of 1:14-40:288-865 respectively. In anembodiment, the liquid pharmaceutical composition comprises adalimumab,histidine buffering agent (or buffer system), sugar stabiliser, and atonicifier in a molar ratio of 1:14-40:288-865:28-576 respectively.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, histidine buffering agent (or buffer system), and a sugarstabiliser in a molar ratio of 1:14-40:548-605 respectively. In anembodiment, the liquid pharmaceutical composition comprises adalimumab,histidine buffering agent (or buffer system), sugar stabiliser, and atonicifier in a molar ratio of 1:14-40:548-605:115-173 respectively.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, histidine (or histidine buffer system), and trehalose in amolar ratio of 1:5.7-145:288-865 respectively. In an embodiment, theliquid pharmaceutical composition comprises adalimumab, histidine (orhistidine buffer system), trehalose, and sodium chloride in a molarratio of 1:5.7-145:288-865:28-576 respectively.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, histidine (or histidine buffer system), and trehalose in amolar ratio of 1:14-40:548-605 respectively. In an embodiment, theliquid pharmaceutical composition comprises adalimumab, histidine (orhistidine buffer system), trehalose, and sodium chloride in a molarratio of 1:14-40:548-605:115-173 respectively.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, histidine (or histidine buffer system), and trehalose in amolar ratio of 1:28.8:576 respectively. In an embodiment, the liquidpharmaceutical composition comprises adalimumab, histidine (or histidinebuffer system), trehalose, and sodium chloride in a molar ratio of1:28.8:576:144 respectively.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, histidine (or histidine buffering species), and trehalose ina weight ratio of 25-75:0.31-7.8:15-140 respectively. In an embodiment,the liquid pharmaceutical composition comprises adalimumab, histidine(or histidine buffering species), trehalose, and sodium chloride in aweight ratio of 25-75:0.31-7.8:15-140:0.5-12 respectively.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, histidine (or histidine buffering species), and trehalose ina weight ratio of 45-55:0.77-2.2:65-72 respectively. In an embodiment,the liquid pharmaceutical composition comprises adalimumab, histidine(or histidine buffering species), trehalose, and sodium chloride in aweight ratio of 45-55:0.77-2.2:65-72:2.7-3.1 respectively.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, histidine (or histidine buffering species), and trehalose ina weight ratio of 50:1.55:68 respectively. In an embodiment, the liquidpharmaceutical composition comprises adalimumab, histidine (or histidinebuffering species), trehalose, and sodium chloride in a weight ratio of50:1.55:68:2.9 respectively.

Any of the aforementioned embodiments relating to molar and/or weightratios of the various components may be additionally defined byreference to the (substantial or entire) absence or low levels ofcomponent(s) such as arginine, amino acids other than histidine,surfactants (optionally with the exception of polysorbate 80), and/orphosphate buffering agents/systems, as defined anywhere herein.

It will be appreciated that the buffering agent (e.g. histidine) orbuffer system (e.g. histidine/imidazolium-histidine) of any of theaforementioned embodiments may be directly incorporated into thecompositions or may be produced in situ, for instance, via an acid basereaction, suitably by reacting a conjugate acid of the buffering agent(e.g. imidazolium form of histidine, whether a pre-formed salt such ashistidine hydrochloride or the imidazolium form generated upondissolution of free histidine) with a base (e.g. sodium hydroxide).Regardless of the method used to provide or produce the buffering agentor buffer system, suitably the resulting composition ultimatelycomprises an appropriate balance of the buffering agent and anyacid/base conjugate to furnish the desired pH. The skilled person willbe readily able to calculate or experimentally determine, without undueeffort, the appropriate balance of buffering agent and acid/baseconjugate, and/or the amount of base which needs to be added to aconjugate acid in order to produce the appropriate amount of bufferingagent and furnish the desired pH.

In an embodiment, the liquid pharmaceutical composition comprises:

-   -   adalimumab;    -   an histidine buffering agent (e.g. histidine) (or histidine        buffer system);    -   a sugar stabiliser (e.g. trehalose);    -   a tonicifier (e.g. sodium chloride);    -   optionally a surfactant (e.g. polysorbate 80); and    -   water (for injection);    -   wherein the composition:        -   is (substantially or entirely) free of arginine (suitably            L-arginine); comprises arginine in a concentration of at            most 0.1 mM;        -   is (substantially or entirely) free of amino acids other            than histidine or comprises one or more amino acids other            than histidine in a (collective) concentration of at most            0.1 mM;        -   is (substantially or entirely) free of surfactants with the            optional exception of polysorbate 80 or comprises one or            more of said surfactants (optionally excluding            polysorbate 80) in a (collective) concentration of at most 1            mM; and/or        -   is (substantially or entirely) free of phosphate buffering            agents (e.g. sodium dihydrogen phosphate, disodium hydrogen            phosphate) or comprises a phosphate buffer system in a            concentration of at most 0.1 mM.

In an embodiment, the liquid pharmaceutical composition comprises:

-   -   Adalimumab (suitably in a concentration as defined herein);    -   5 to 14 mM histidine buffering agent (e.g. histidine) (or        histidine buffer system);    -   100 to about 300 mM a sugar stabiliser (e.g. trehalose);    -   10 to about 200 mM a tonicifier (e.g. sodium chloride); and    -   water (for injection);    -   wherein the composition:        -   has a pH between 6.3 and 6.7 (e.g. pH 6.4)        -   is (substantially or entirely) free of arginine; comprises            arginine in a concentration of at most 0.1 mM;        -   is (substantially or entirely) free of amino acids other            than histidine or comprises one or more amino acids other            than histidine in a (collective) concentration of at most            0.1 mM;        -   is (substantially or entirely) free of surfactants with the            optional exception of polysorbate 80 or comprises one or            more of said surfactants (optionally excluding            polysorbate 80) in a (collective) concentration of at most 1            mM; and/or        -   is (substantially or entirely) free of phosphate buffering            agents (e.g. sodium dihydrogen phosphate, disodium hydrogen            phosphate) or comprises a phosphate buffer system in a            concentration of at most 0.1 mM.

In an embodiment, the liquid pharmaceutical composition comprises:

-   -   25 to about 75 mg/mL adalimumab;    -   2 to about 50 mM histidine (or histidine buffer system);    -   100 to about 300 mM trehalose;    -   10 to about 200 mM sodium chloride; and    -   water (for injection);    -   wherein the composition:        -   has a pH between 6.3 and 6.5;        -   is (substantially or entirely) free of arginine (suitably            L-arginine) or comprises arginine in a concentration of at            most 0.1 mM;        -   is (substantially or entirely) free of amino acids other            than histidine or comprises one or more amino acids other            than histidine in a (collective) concentration of at most            0.1 mM;        -   is (substantially or entirely) free of surfactants or            comprises one or more surfactants in a (collective)            concentration of at most 1 mM; and/or        -   is (substantially or entirely) free of phosphate buffering            agents (e.g. sodium dihydrogen phosphate, disodium hydrogen            phosphate) or comprises a phosphate buffer system in a            concentration of at most 0.1 mM.

In an embodiment, the liquid pharmaceutical composition comprises:

-   -   45 to about 55 mg/ml adalimumab;    -   5 to 14 mM histidine (or histidine buffer system);    -   190 to 210 mM trehalose;    -   40 to 60 mM sodium chloride; and    -   water (for injection);    -   wherein the composition:        -   has a pH between 6.3 and 6.5;        -   is (substantially or entirely) free of arginine (suitably            L-arginine) or comprises arginine in a concentration of at            most 0.001 mM;        -   is (substantially or entirely) free of amino acids other            than histidine or comprises one or more amino acids other            than histidine in a (collective) concentration of at most            0.001 mM.        -   is (substantially or entirely) free of surfactants or            comprises one or more of surfactants in a (collective)            concentration of at most 0.0001 mM; and/or        -   is (substantially or entirely) free of phosphate buffering            agents (e.g. sodium dihydrogen phosphate, disodium hydrogen            phosphate) or comprises a phosphate buffer system in a            concentration of at most 0.001 mM.

In an embodiment, the liquid pharmaceutical composition comprises:

-   -   50 mg/ml adalimumab;    -   10 mM histidine (or histidine buffer system);    -   200 mM trehalose;    -   50 mM sodium chloride;    -   1.0 mg/mL polysorbate 80; and    -   water (for injection);    -   wherein the composition:        -   has a pH of 6.4;        -   is free of arginine;        -   is free of amino acids other than histidine;        -   is free of surfactants; and        -   is free of phosphate buffering agents/buffer systems.

Preferably, the liquid pharmaceutical composition consists essentiallyof:

-   -   25 to about 75 mg/mL adalimumab;    -   2 to about 50 mM histidine (or histidine buffer system);    -   100 to about 300 mM trehalose;    -   10 to about 200 mM sodium chloride; and    -   water (for injection);        -   wherein the composition has a pH between 6.3 and 6.5.

Preferably, the liquid pharmaceutical composition consists essentiallyof:

-   -   40 to about 60 mg/mL adalimumab;    -   5 to about 15 mM histidine (or histidine buffer system);    -   175 to about 225 mM trehalose;    -   25 to about 75 mM sodium chloride; and    -   water (for injection);        -   wherein the composition has a pH between 6.3 and 6.5.

Preferably, the liquid pharmaceutical composition consists essentiallyof:

-   -   50 mg/mL adalimumab;    -   10 mM histidine (or histidine buffer system);    -   200 mM trehalose;    -   50 mM sodium chloride; and    -   water (for injection);        -   wherein the composition has a pH of 6.4.

Suitably, the liquid pharmaceutical composition may be as set forth inany of the preceding embodiments, except that the absence or low levelsof component(s) such as arginine, amino acids, surfactants (optionallywith the exception of polysorbate 80), and phosphate bufferingagents/systems, rather than being defined by reference toconcentration(s) (i.e. molarity) may instead be defined by reference tocorresponding molar ratios of the component to buffering agent/buffersystem; corresponding weight ratios of the component to adalimumab; orcorresponding molar ratios of the component to adalimumab. The skilledperson will readily deduce for each component, from the relevant sectionof this specification relating to that specific component, which molarand weight ratios correspond to which concentrations, since herein therelevant molar and weight ratios are listed to respectively correspondto given concentrations. For example, in the case of arginine, theoptional concentrations of “at most 0.1 mM, more suitably at most 0.01mM, most suitably at most 0.001 mM” respectively correspond with a molarratio of arginine to buffering agent of “at most 1:150 . . . moresuitably at most 1:1500, most suitably at most 1:15,000”; with “a weightratio of arginine to adalimumab of at most 1:3000 . . . more suitably atmost 1:30,000, most suitably at most 1:300,000”; and with a molar ratioof arginine to adalimumab of at most 1:3.75 . . . more suitably at most1:37.5, most suitably at most 1:375″. The same correspondences apply foramino acids, surfactants, and phosphate buffering agents/systems.

Method of Manufacturing a Liquid Pharmaceutical Composition

The present invention provides a method of manufacturing a liquidpharmaceutical composition, suitably as defined herein. The methodsuitably comprises mixing together, in any particular order deemedappropriate, any relevant components required to form a liquidpharmaceutical composition as defined herein. The skilled person mayrefer to the Examples or techniques well known in the art for formingliquid pharmaceutical compositions (especially those for injection viasyringe). Different embodiments will suitably require differentcombinations of components to be mixed, potentially in differentamounts. The skilled person can readily deduce such combinations andamounts by reference to the foregoing disclosure relating to the liquidpharmaceutical composition.

Suitably the method involves mixing together the relevant componentssuitably, in a diluent (e.g. water), suitably so that all of thecomponents are (substantially or entirely) dissolved in the diluent.

The method may involve first preparing a pre-mixture (or pre-solution)of some or all components (optionally with some or all of the diluent)excluding adalimumab, and adalimumab may then itself (optionally with orpre-dissolved in some of the diluent) be mixed with the pre-mixture (orpre-solution) to afford the liquid pharmaceutical composition, or acomposition to which final components are then added to furnish thefinal liquid pharmaceutical composition. Most suitably, the pre-mixturecontains all components except for the adalimumab and optionally alsosome diluent (which may be used to pre-dissolve adalimumab), suitably sothat adalimumab is added to a mixture which offers optimal stabilisationof adalimumab. Suitably the aforementioned pre-mixture is prepared withthe desired pH for the final liquid pharmaceutical formulation.

Suitably, the method involves forming a buffer system, suitably a buffersystem comprising a buffering agent as defined herein. The buffer systemis suitably formed in a pre-mixture prior to the addition of adalimumab,though the buffer system may optionally be formed with adalimumabpresent. The buffer system may be formed through simply mixing thebuffering agent (supplied ready-made) with its acid/base conjugate(suitably in appropriate relative quantities to provide the desiredpH—this can be determined by the skilled person either theoretically orexperimentally). In the case of an histidine buffer system, this meansmixing histidine with an imidazolium form of histidine (e.g. histidinehydrochloride). Alternatively, the buffer system may be formed throughadding a strong acid (e.g. HCl) to the buffering agent (e.g. histidine)in order to form in situ the acid/base conjugate (e.g. imidazolium formof histidine) to the buffering agent (again suitably in appropriaterelative quantities to provide the desired pH). Alternatively, thebuffer system may be formed through adding a strong base (e.g. sodiumhydroxide) to the acid/base conjugate of the buffering agent (or to thebuffering agent itself, where it forms said acid/base conjugate in adynamic equilibrium, such as upon dissolution) in order to form in situthe buffering agent (again suitably in appropriate relative quantitiesto provide the desired pH). The pH of either the pre-mixture of finalliquid pharmaceutical composition may be judiciously adjusted by addingthe required quantity of strong base or strong acid, or even a quantityof buffering agent or acid/base conjugate.

In certain embodiments, the buffering agent and/or buffer system ispre-formed as a separate mixture, and the buffer system is transferredto a precursor of the liquid pharmaceutical composition (comprising someor all components save for the buffering agent and/or buffer system,suitably comprising adalimumab and potentially only adalimumab) viabuffer exchange (e.g. using diafiltration until the relevantconcentrations or osmolality is reached). Additional excipients may beadded thereafter if necessary in order to produce the final liquidpharmaceutical composition. The pH may be adjusted once or before allthe components are present.

Any, some, or all components may be pre-dissolved or pre-mixed with adiluent prior to mixing with other components.

The final liquid pharmaceutical composition may be filtered, suitably toremove particulate matter. Suitably filtration is through filters sizedat or below 1 μm, suitably at 0.22 μm. Suitably, filtration is througheither PES filters or PVDF filters, suitably with 0.22 μm PES filters.

The present invention also provides a liquid pharmaceutical compositionobtainable by, obtained by, or directly obtained by the method ofmanufacture herein described.

Drug-Delivery Device

The present invention provides a drug delivery device comprising aliquid pharmaceutical composition as defined herein. Suitably the drugdelivery device comprises a chamber within which the pharmaceuticalcomposition resides. Suitably the drug delivery device is sterile.

The drug delivery device may a vial, ampoule, syringe, injection pen(e.g. essentially incorporating a syringe), or intravenous bag. Mostsuitably the drug delivery device is a syringe, suitably an injectionpen. Suitably the syringe is a glass syringe. Suitably the syringecomprises a needle, suitably a 29G ½″ needle.

The present invention provides a method of manufacturing a drug deliverydevice, suitably as defined herein, the method comprising incorporatinga liquid pharmaceutical composition as defined herein within a drugdelivery device. Such manufacture typically involves charging the liquidpharmaceutical composition as defined herein to a syringe, suitably viaa needle affixed thereto. The needle may thereafter be removed,replaced, or remain.

According to an eleventh aspect of the present invention there isprovided a drug delivery device obtainable by, obtained by, or directlyobtained by a method of manufacture defined herein.

Package

The present invention provides a package comprising a liquidpharmaceutical composition as defined herein. Suitably the packagecomprises a drug delivery device as defined herein, suitably a pluralityof drug delivery devices. The package may comprise any suitablycontainer for containing one or more drug delivery devices.

The present invention provides a method of manufacturing a package, themethod comprising incorporating a liquid pharmaceutical composition asdefined herein within a package. Suitably this is achieved byincorporating said liquid pharmaceutical composition within one or moredrug delivery devices, and thereafter incorporating the one or morepre-filled drug delivery devices into a container present within thepackage.

The present invention provides a package obtainable by, obtained by, ordirectly obtained by a method of manufacture defined herein.

Kit of Parts

The present invention provides a kit of parts comprising a drug deliverydevice (without the liquid pharmaceutical composition incorporatedtherein), a liquid pharmaceutical composition as defined herein(optionally contained in a separate package or container), andoptionally a set of instructions with directions regarding theadministration (e.g. sub-cutaneous) of the liquid pharmaceuticalcomposition. The user may then fill the drug delivery device with theliquid pharmaceutical composition (which may be provided in a vial orampoule or such like) prior to administration.

Uses of Pharmaceutical Liquid Composition and Methods of Treatment

According to a twelfth aspect of the present invention there is provideda method of treating a disease or medical disorder; a liquidpharmaceutical composition for use in therapy; a use of a liquidpharmaceutical composition in the manufacture of a medicament for thetreatment of a disease or disorder; a method of treating a tumournecrosis factor-alpha (TNF-α)-related autoimmune disease; a liquidpharmaceutical composition for use in the treatment of a tumour necrosisfactor-alpha (TNF-α)-related autoimmune disease; a use of a liquidpharmaceutical composition in the manufacture of a medicament for thetreatment of a tumour necrosis factor-alpha (TNF-α)-related autoimmunedisease; a method of treating rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, Crohn's disease, ulcerative colitis, moderate tosevere chronic psoriasis and/or juvenile idiopathic arthritis; a liquidpharmaceutical composition for use in the treatment of rheumatoidarthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease,ulcerative colitis, moderate to severe chronic psoriasis and/or juvenileidiopathic arthritis; and a use of a liquid pharmaceutical compositionin the manufacture of a medicament for the treatment of rheumatoidarthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease,ulcerative colitis, moderate to severe chronic psoriasis and/or juvenileidiopathic arthritis; as defined herein.

The liquid pharmaceutical compositions defined herein may be used totreat any one or more of the aforementioned diseases or medicaldisorders. In a particular embodiment, the liquid pharmaceuticalcompositions are used to treat rheumatoid arthritis, suitably Crohn'sdisease and psoriasis.

The liquid pharmaceutical compositions are suitably parenterallyadministered, suitably via sub-cutaneous injection.

EXAMPLES Materials and Equipment

The following materials were used in the preparation of formulationsdescribed in the Examples that follow:

The following disposable equipment and materials were used in theExamples and Screen Experiments which follow.

Item Code Supplier Eppendorf Tubes NA Eppendorf (0.5 mL, 1.5 mL, 2.0 mL)Falcon 352096 (15 mL), 352070 Becton (50 mL) polypropylene tubesDickinson PES membrane (0.22 μm) MillexGP Express PES Millipore filterunit membrane REF SLGP033RS PETG bottles 3420-1000, 3420-0500, Nalgene2019-0250, 3420-0125, 3420-0060, 2019-0030

The following packaging was used in the Examples and Screen Experimentswhich follow.

Item Code Supplier DIN2R Type I glass vial 0212060.6112 11200000A NuovaOmpi 1 mL stopper S2-F451 RSV; Daikyo Seiko, D 21-7S RB2-40 LTD 13 mmflip-off cap 12000350 MS-A

The following equipment was used in the Examples and Screen Experimentswhich follow.

Item Mod. Manufacturer Analytical scales AX205, PG2002-S Mettler ToledoBenchtop xenon Suntest CPS+ Atlas instrument Calibrated pipettes P20,P100, P200, P1000 Gilson HPLC Alliance Waters iCE280 Fast IEF AnalyzerConvergent Bioscience Osmometer Osmomat 030/D Gonotec PCR 7500 FastReal-Time AB Applied Biosystem pH meters Seven Multi Mettler ToledoRefrigerators +2-8° C. Angelantoni Software Design ver. 7.1.5 Stat-Ease,Inc. Expert Thermostatic cabinets +25° C., +40° C. AngelantoniTurbidimeter 2100AN IS Hach Lange UV Spectrophotometer Lambda 35 PerkinElmer

Analytical Techniques and Protocols

The following analytical methods of protocols were employed, in theExamples and Screening Experiments which follow, for the reasons statedin the table below:

Method No. Analytical Method Scope of the test 1 Bioanalyzer Purity 2DSF Unfolding temperature 3 iCE280 Isoforms profile 4 OD Protein Content5 SE-HPLC Aggregates determination 6 Nephelometry Turbidity 7 OsmolalityOsmolality of solution 8 pH pH determination 9 Sub-visible particlesParticle count

The individual protocols for each of the above analytical methods aredescribed in turn below, and references in the Examples and ScreeningExperiments to any such analytical methods used these protocols.

1. Purity—Bioanalyzer

A 2100 Bioanalyzer was used. Protocols can be found in the relevantinstruction manual. However, the protocols have been additionallyrefined as follows.

Solutions:

Gel-Dye Mix (Staining solution):

Add 25 μL of 230 plus dye concentrate to a protein 230 plus gel matrixtube. Vortex well, and spin down the tube for 15 seconds. Transfer to aspin filter and centrifuge at 2500 rpm for at least 20 min. The solutionis ready to use. Store the solution at +5±3° C. for not more than 4weeks.

Destaining Solution:

Pipette 650 μL of gel matrix into a spin filter. Centrifuge at 2500 rpmfor at least 25 min. Store the solution at +5±3° C. for not more than 4weeks.

Sample Buffer:

It is recommended to divide the 200 μL of sample buffer into aliquots of25 μL and defreeze aliquot for each chip. Store the Sample buffer stocksolution and the aliquots at −20° C., not longer than the expiring dateprovided by the supplier.

Maleimide Stock Solution:

Dissolve 23.4 mg of Male imide in I mL MilliQ water (0.24M). Vortex wellthe solution. Subsequently dilute the solution 1:4 with MilliQ water.(e.g. 50 μL Stock Sol.+150 μL MilliQ). The final concentration of thediluted Maleimide solution is 60 mM. (Since no data is available yet onthe stability of this solution, it must be prepared freshly beforestarting each analytical session).

OTf-Solution:

For the analysis of Adalimurnab samples the reducing solution must beprepared with 1M DTT, therefore dissolve 154.0 mg DTT in 1 mL MilliQWater.

Non-Reducing Solution:

Add 1 μl, of MilliQ water to a sample buffer aliquot (25 μL) and vortexfor 5 seconds. Use the non-reducing solution within its preparation day.

Reducing Solution:

Add 1 μl, of the according DTf-Solution to a sample buffer aliquot (25μL) and vortex for 5 seconds. Use the reducing solution within itspreparation day.

Sample Preparation:

-   -   Samples are analyzed at the concentration ranging between 2.4-3        mg/ml.    -   If it's necessary the samples can be diluted to the target        concentration using Milli Q water.

Samples are prepared according to the Reagent Kit Guide using thereducing and non reducing sample buffers according to the instruction inthe Reagent Kit Guide and also mentioned above. It is stronglyrecommended to use, differently from the guide, greater volumes toachieve reproducible and accurate results. An example how to prepare theladder and the samples is reported below:

Sample preparation solution Reducing and Non Reducing condition. TotalReagent Volume μl Volume μL Sample diluted at 3 mg/ml 3 μL 6 μL Samplebuffer (reducing or 2 μL not reducing) Maleimide solution 1 μL Sampleshave to be mixed (via vortex) well and spinned down All Samples and theLadder are heated 5 minutes to 70° C. MilliQ water 84 μL  90 μL  vortexwell and spin down Loading 6 μL all Sample and Ladder Note 1: For theIPCs whose concentration is between 2.4 mg/ml and 3.0 mg/mL, the samplepreparation follows the table above but the volume of MilliQ Water addedafter sample heating is calculated in order to reach a final proteinconcentration of 0.1 mg/ml.

An example of sample preparation for a sample having concentrationbetween 2.4 and 3.0 mg/mL, is reported here below:

Sample preparation solution Reducing and Non Reducing condition TotalReagent Volume μl Volume μL Sample (2.6 mg/mL) 3 μL 6 μL Sample buffer(reducing or 2 μL not reducing) Maleimide soluton 1 μL Samples have tobe mixed (via vortex) well and spinned down All Samples and the Ladderare heated 5 minutes at 70° C. MilliQ water 72 μL  78 μL  vortex welland spin down Loading 6 μL all Sample and Ladder Note 2: All wells haveto be loaded. If the sample number is lower than the available wells,the empty wells can be used for additional duplicates or blank samples.

Preparing the System and the Chip:

-   -   To clean the system before and as well after an analysis fill        the “Electrode Cleaner” with 600 IIL MilliQ Water and place it        into the Agilent 2100 Bioanalyzer, close the lid and let the        system cease. No further action is required.    -   Adjust the base-plate of the chip priming station to position        “A” and the syringe clip to its middle position.

Preparing the chip System Preparation Insert a new Protein Chip in thepriming station Pipette 12 μL of Gel-Dye Mix in the well marked G (Upright) Set the plunger at 1 mL and close the chip priming station Pressthe plunger until it is held by the clip Wait 60 seconds and thenrelease the clip Wait 5 seconds and slowly pull back the plunger to the1 mL mark Open the chip priming station Remove the solution in this wellPipette 12 μL of Gel-Dye Mix into the well marked G (UP right) and inall remaining wells marked with G Pipette 12 μL of Destaining Solutionin the well marked with DS

Loading of the Ladder and the Sample:

-   -   Transfer 6 μL of each sample into a sample well and as well 6 μL        of the ladder in the dedicated well, which is clearly indicated        with a ladder symbol.

Place the chip into the Agilent 2100 Bioanalyzer and start the analysiswithin 5 min.

Example of sample set Well Sample Amount μL 1 Blank 6 2 Blank 6 3Unknown sample1 rep1 6 4 Unknown sample1 rep2 6 5 Unknown sample2 rep1 66 Unknown sample2 rep2 6 7 Unknown sample3 rep1 6 8 Unknown sample3 rep26 9 Current Reference Material rep1 6 10 Current Reference Material rep26 Ladder Ladder 6

Data Analysis and Evaluation of the Results:

To gain results the following minimum operations have to be executed

-   -   Place the chip in the specific spot and close the lid.    -   In the instrument context select        Assay-Electrophoresis-Protein-Protein 230 Plus.    -   Click on START to start the analysis, which is completed within        30 minutes.    -   The raw data are shown by clicking on “Data Analysis” where all        experiments, carried out at the day, are listed. Click on the        experiment of interest and select it.    -   The gel generated from the chosen experiment is automatically        opened.    -   Data can be shown as an electropherogram or gel-like image.

Detailed information regarding the integration of the peaks in theelectropherogram (to gain the purity data) is included in the manual ofthe software. The purity of a sample is automatically given by thesystem by automatic integration, but if needed, the manual integrationcan be applied.

Results:

In non reducing condition the results are indicated as % Purity, and %LMW (sum of peaks before monomer).

In reducing condition the results are indicated as % Purity, as sum ofheavy chain and light chain.

The indicative molecular weight values are reported in the table below:

Indicative molecular weight of Adalimumab Condition Results KDa NonReducing Monomer 151 Reducing LC 27 HC 58

2. Unfolding Temperature—DSF

DSF (differential scanning fluorimetry) was performed as follows:

2 microliters of Sypro Orange (Orange protein gel stain, cod. S6650,Life Technologies) previously diluted 500-fold in water for injectionwere added to 20 microliters of drug product solution. Upon addition ofSypro Orange, the DP solutions (triplicate preparation) are filled in96-well plates (MicroAmp Fast 96-W Reaction Plate 0.1 mL, cod. 4346907).The plates are then sealed with a protective, transparent cover(MicroAmp Optical Adhesive Film, cod. 4311971) and then subjected tocentrifugation to remove air bubbles. The plates are then inserted inthe 7500 Fast Real-Time AB Applied Biosystem PCR system and scanned foremission profiles at temperatures from room temperature to 90-100° C.The dependence of intensity of fluorescence emission on temperature is acurve which typically shows an inversion point/discontinuation at thedenaturation temperature, parameter used to compared the differentcompositions.

3. Isoforms Profile—iCE280

cIEF by iCE280 (isoforms profile): After purification and removal ofsalts with centrifugation in Amicon Ultra-4 centrifugal devices (cut off10 kDa), the samples were pre-diluted to the concentration of 5.0 mg/mLwith purified water. A second dilution was then made to 1.0 mg/mL with asolution composed of: methyl cellulose, Pharmalyte 5-8 (GE Healthcare),Pharmalyte 8-10.5 (GE Healthcare), low pl marker 7.05 (Protein Simple),high pl marker 9.50 (Protein Simple) and purified water. Upon dilutionthe samples were centrifuged at 10000 rpm for 3 minutes. An additionalcentrifugation step 2 minutes at 7000 rpm) is then conducted on 150microL of each sample transferred in glass inserts. The cIEF (capillaryisoelectric focusing) was carried out with the iCE280 system by ProteinSimple, using capillary cartridges Fc with 100 micron ID coating andtotal length of 50 nm (Cat. No. 101700/101701 by Protein Simple). Theseparation of the various isoforms is made using 100 mM sodium hydroxide(in 0.1% methyl cellulose) as a cathodic solution and 80 mM o-phosphoricacid (in 0.1% methyl cellulose) as an anodic solution. Theelectropherogram is acquired at 280 nm over pre-focusing and focusingtimes of 1 and 6 minutes respectively, at a voltage of 1500 V(pre-focusing) and 3000 V (focusing).

4. Protein Content—OD

OD (protein content) measurements were taken on samples which wereinitially diluted gravimetrically (triplicate independent dilutions weremade) with relevant buffer or placebo from starting concentration toabout 10 mg/mL. The diluted solutions were tested for absorbance at 280and 320 nm in 0.1 cm pathlength quartz cuvettes, at room temperature,with a double-beam spectrophotometer (Lambda 35 by Perkin Elmer). Thevalue of 1.35 was used as molar extinction coefficient of Adalimumab.

5. Aggregates Determination—SE-HPLC

The samples were diluted with DPBS 1× to a concentration of 0.5 mL andinjected (20 microL injection volume) in a Column TSK gel Super SW30004.6 mm ID X30.0 cm cod.18675 by Tosoh maintaining isocratic conditions(mobile phase: 50 mM Sodium Phosphate+0.4M Sodium perchlorate, pH6.3±0.1). UV detection was made at 214 nm at a flow rate of 0.35 mL. Theduration of each analytical run was 15 minutes. Prior to the analysisthe samples were maintained at 2-8° C. in the autosampler of the WatersAlliance HPLC systems used for this test.

6. Turbidity—Nephelometry

Turbidity was assessed via nephelometric (effect based on the lightdiffusion effect caused by particles with dimensions typically <1micron) measurements conducted with a turbidimeter 2100AN ISTurbidimeter by Hach at room temperature. Minimum amounts of 3 mL ofsolution were placed in reduced volume glass cuvettes and tested fordiffusive effect after prior calibration of the instrument with a seriesof standard solutions (0.1-7500 NTU).

7. Osmolality Determination—Osmolality

Osmolality was measured based on the cryoscopic characteristic of thesolutions. The tests were conducted with an Osmomat 030-D by Gonotechsubjecting 50 microL of the sample to freezing. The freezing temperaturedepends on the osmolality of the solution (i.e. on the presence ofagents dissolved such as salts, sugars, other ionic and non ionicspecies, etc).

8. pH Determination—pH

pH was determined using potentiometric measurements conducted at roomroom temperature with Mettler Toledo Seven Multi pH meters.

9. Particle Count—Sub-Visible Particles

The samples were 5-fold diluted with purified water to a final volume of25 mL. The number of particles are determined at room temperature byPAMAS SVSS by Aminstruments collecting four independent runs andaveraging the results for each respective dimensional fraction ofinterest.

Example 1—Formulations for First Formulation Screen

The following first set of formulations (often referenced herein as DoE1formulations) are shown below in Table 1.

TABLE 1 List of DoE1 formulations for later Screen Experiments 1 Salt(NaCl) Buffer Form conc type # (mM) (10 mM) pH Stabilizer 18 25Histidine 6.0 Trehalose dihydrate (200 mM) 19 50 Histidine 6.0 LysineHydrochloride (100 mM) 20 100 Histidine 6.0 Mannitol (200 mM) 21 50Histidine 6.2 Lysine Hydrochloride (100 mM) 22 50 Histidine 6.2 ArginineMonohydrocloride + Aspartic Acid (80 mM + 20 mM) 23 75 Histidine 6.2Trehalose dihydrate (200 mM) 24 25 Histidine 6.4 Mannitol (200 mM) 25100 Histidine 6.4 Trehalose dihydrate (200 mM)

The formulations of Table 1 were manufactured starting from apreformulated, surfactant-free DS material.

An aliquot of the DS has been diafiltrated with 10 mM histidine bufferat pH 6.0 until a three-fold volume exchange with the buffer wasachieved. Then the required excipients have been added to thebuffer-exchanged DS materials and the pH adjusted to the target byaddition of a diluted solution of sodium hydroxide. Each formulation wasfiltered through 0.22 μm PES filters.

In Table 2, the results in terms of material recovery and osmolality forthe three buffer-exchanges DS materials are reported.

There was good recovery for the histidine buffer system (≦90%). Theosmolality values indicate the satisfactory degree of buffer exchangereached, with a minimal residual of species coming from the originatingDS.

Example 2—Formulations for Second Formulation Screen

The following second set of formulations (often referenced herein asDoE2 formulations) are shown below in Table 3 (as derived from Table 4below that).

TABLE 3 List of DoE2 formulations for later Screen Experiments 2(formulations derived from that presented in Table 4 with the extrasurfactant indicated) Polysorbate 80 concentration (mg/mL) Formulations0 0.5 1 Form 7 (deriving from Form C, Table 4) X — — Form 8 (derivingfrom Form C, Table 4) — x — Form 9 (deriving from Form C, Table 4) — — x

TABLE 4 Formulation prototype deriving from the DoE1 screen Salt (NaCl)Buffer type Form mM (10 mM) pH Stabilizer C 100 Histidine 6.4 Trehalosedihydrate (200 mM)

The DoE2 formulations (Table 3) were manufactured starting from apreformulated, surfactant-free, DS material.

Three aliquots of the DS have been diafiltrated until a three-foldvolume exchange was achieved. Then the required excipients have beenadded to the buffer-exchanged DS materials and the pH adjusted to thetarget by addition of a diluted solution of sodium hydroxide. Eachformulation was filtered through 0.22 μm PES filters.

In Table 5, the results in terms of osmolality and turbidity for thebuffer-exchanges DS materials are reported.

The osmolality values (≦40 mOsm/kg) indicated the satisfactory degree ofbuffer exchange reached, with a minimal residual of species coming fromthe originating DS.

TABLE 5 Osmolality and turbidity of the DS materials after bufferexchange Turbidity Osmolality Buffer (NTU) (mOsm/kg) Histidine 50 26

Example 3—Comparative Formulations for Both First and Second Screens

For comparison and control purposes, three reference formulations wereprepared or obtained, including Ref-1 (Humira® composition manufacturedby the Applicant); Ref-2 (RMP US—Humira® commercial drug product fromthe USA); and Ref-3 (RMP EU—Humira® commercial drug product from theEU). All of these reference formulations had the composition shown inTable 6.

TABLE 6 Composition of Humira DP Amount per container (mg) AmountIngredient (filling volume = 0.8 mL) (mg/mL) Adalimumab 40 50 CitricAcid Monohydrate 1.04 1.3 Dibasic sodium phosphate 1.22 1.53 dehydrateMannitol 9.6 12 Monobasic sodium phosphate 0.69 0.86 dehydratePolysorbate 80 0.8 1 Sodium chloride 4.93 6.16 Sodium citrate 0.24 0.3WFI and sodium hydroxide q.b. to adjust q.b. to adjust pH to 5.2 pH to5.2

Screening

A first formulation screen (DoE1) led to the identification of variousfactors (e.g. pH, presence of NaCl, excipient type) responsible forprotein stability, and ultimately to the selection of formulations to bepursued in a second screen (DoE2), which sought to fine-tune theformulations and evaluate how surfactants, such as Polysorbate 80, canimpact the stability of the protein.

Each of the two screens involved various analytical testing, as definedhereinbefore and referred to hereinafter, upon a range of differentformulations which were exposed to varying levels of thermal,mechanical, and light stress over prolonged periods (e.g. 1 month).These formulation screens enabled the gathering of a significant amountof data, which provided surprising and valuable insights allowing forthe development of new advantageous formulations.

The results of the two formulations screens are presented below.

Screening Experiment 1—Analysis and Screening of Example 1 FormulationsAgainst Comparative Formulations of Example 3

Preliminary DoE screening (Step 1) evaluated the effect that ionicstrength (given by NaCl), pH and different stabilizers exerts on theprotein in the course of short term stability studies.

A response surface D-Optimal statistical design has been applied. Threefactors were considered:

-   -   Ionic strength (driven by NaCl concentration, which was varied        in the range 25 mM-100 mM and was set as a numeric factor),    -   pH (the range 4.6-6.4) buffered by histidine was investigated;    -   Stabilizer/Excipient (categoric factor comprising several        levels: Lysine Hydrochloride, Arginine+Aspartic Acid, Mannitol,        Trehalose Dihydrate).

These formulations were manufactured, as described in Example 1 above,starting from DS without Polysorbate 80 and were thereforesurfactant-free.

Table 7 below summarizes the formulations tested within this screening.In addition to the 8 formulations proposed, two controls have also beenanalyzed as comparators:

-   -   Humira commercial drug product DP (Formulated as per Example 3        above)    -   MS drug substance DS formulated as Humira commercial DP        (Formulated as per Example 3 above)

TABLE 7 List of DoE1 formulations (Step 1) screened through thermalstress conditions (stability at 40° C.) and high throughputdetermination of protein unfolding temperature (DSF). Salt (NaCl) BufferForm conc type # (mM) (10 mM) pH Stabilizer 18 25 Histidine 6.0Trehalose dihydrate (200 mM) 19 50 Histidine 6.0 Lysine Hydrochloride(100 mM) 20 100 Histidine 6.0 Mannitol (200 mM) 21 50 Histidine 6.2Lysine Hydrochloride (100 mM) 22 50 Histidine 6.2 ArginineMonohydrocloride + Aspartic Acid (80 mM + 20 mM) 23 75 Histidine 6.2Trehalose dihydrate (200 mM) 24 25 Histidine 6.4 Mannitol (200 mM) 25100 Histidine 6.4 Trehalose dihydrate (200 mM) Ref-1 Humira composition(formulation manufactured (MS) with MS Drug Substance) - Example 3 Ref-2Humira commercial DP (USA) - Example 3 (RMP US) Ref-3 Humira commercialDP (EU) - Example 3 (RMP EU)

The formulations were tested according to the plan reported in Table 8.Thermal stress up to 1 month at 40° C. was considered. High throughputassessment made with the DSF technique (aimed at a fast screening basedon determination of protein unfolding temperature) was performed at TO.

1.1 Osmolality Screen

The osmolality of the DoE1 formulations compounded starting from thebuffer exchanges DS materials (par. 5.1.1) is reported in Table 9.

Most formulations were found in the range of osmolality of 250-400mOsm/kg, while slightly higher values were observed at the highestsodium chloride concentrations.

1.2 Protein Content (OD)

The protein content of the DoE1 formulations was determined at time 0and after 1 month at 40° C.

FIG. 1 is a bar chart showing the protein content (mg/mL) of the DoE1formulations (of Example 1), along with reference standards(representing comparator HUMIRA® formulations), at an arbitrary startpoint (blue bars, time=0) and after 4 weeks (red bars) of theformulation(s) being heated at 40° C.

The results presented in FIG. 1, indicated no significant changesoccurring over time. All concentrations were found in line with thetarget of 50 mg/mL.

1.3 Aggregation (SE-HPLC)

FIG. 2 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE1 formulations (of Example 1), along with referencestandards (representing comparator HUMIRA® formulations), at anarbitrary start point (blue bars, time=0) and after both 2 weeks (greenbars) and 4 weeks (orange bars) of the formulation(s) being heated at40° C. The total aggregates observed by SE-HPLC over stability at 40° C.are graphically represented in FIG. 2. Minimal increases in aggregationwere observed in all formulation. However, even after 1 month, allaggregation levels amounted to less than 1%.

1.4 Fragmentation (Bioanalyzer)

FIG. 3 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE1 formulations (of Example 1), along withreference standards (representing comparator HUMIRA® formulations), atan arbitrary start point (dark blue bars, time=0) and after both 2 weeks(pink bars) and 4 weeks (light blue bars) of the formulation(s) beingheated at 40° C.

In FIG. 3, the variation of fragments over time as determined byBioanalyzer is reported. Formulations at more acidic pH tend to undergofaster fragmentation rates. Moreover, the presence of aminoacids at thispH range can considerably worsen the stability profile.

At pH >6.0 and in presence of sugar/polyols, all the formulas, includingthe references, are comparable (fragmentation lower than 1% after 1month at 40° C.).

Sodium chloride was not found to be a factor critical to stability inthe range 25-100 mM.

1.5 pH Screening

Table 10 shows the pH of the DoE1 formulations (of Example 1), alongwith reference standards (representing comparator HUMIRA® formulations),at an arbitrary start point (time=0) and after both 2 weeks and 4 weeksof the formulation(s) being heated at 40° C.

As can be seen from Table 10, no deviations from targeted pH wereobserved.

1.6 Unfolding Temperature (DSF)

DSF is a high throughput method which aims at the determination of theunfolding temperature of proteins by virtue of increasing interactionswith fluorescent probes as temperature ramps are applied to the samples.When the protein starts to unfold, it will progressively exposehydrophobic patches to the solvent attracting the fluorescent probesthat will pass from the free state in solution (non fluorescent) to thebound state (via hydrophobic interactions) with the protein, thusincreasing the degree of fluorescent signal.

From the evaluation of the fluorescence signal, it was possible todetermine the midpoint of the sigmoidal curves, which indicates thetransition point of each formulation. It is assumed that the higher thetransition point, the higher the resistance of the formula to thermalstress.

The results of the assessment conducted on the DoE1 screeningformulations are reported in FIG. 4. FIG. 4 is a bar chart showing theunfolding temperature (° C.), as determined by DSF, of the DoE1formulations (of Example 1), along with reference standards(representing comparator HUMIRA® formulations).

The unfolding temperature of the three reference formulations is 71-72°C. Few formulations, aside from the references, were found to haveunfolding temperatures higher than 70° C., but those that did include:

-   -   Formulations 23, 24 and 25 (formulations in histidine buffer pH        6.2-6.4 in presence of either Trehalose dihydrate or D-Mannitol        at varying sodium chloride concentrations).

Therefore, this test confirmed the results previously obtained forfragmentation by Bioanalyzer: polyols/sugars can positively impact thethermal stability of the protein, especially at pH ≧6.2, while sodiumchloride does not seem to significantly affect its behavior.

1.7 Iso Forms Profile Change Vs RMP

The isoforms profile of DoE screening formula 25 has been tested after10-11 weeks at 40° C. and compared to Reference samples.

The data, in terms of main peak and acidic cluster variations, arereported in Table 11.

Comparable variations are obtained for the four samples tested, withslightly better performance exhibited by Formulation 25 (in histidine).

TABLE 11 Isoforms profile by iCE280 of most promising formulations fromDoE screening 1 and references. ID Time 0 10 weeks (40° C.) 11 weeks(40° C.) Main DoE1-25 56.5 — 42.2 Ref-1 (MS) 55.8 38.5 — Ref-3 RMP (EU)56.5 40.7 — Ref-2 RMP (US) 56.8 40.6 — Acidic cluster DoE1-25 19.5 —36.9 Ref-1 (MS) 19.8 40.5 — Ref-3 RMP (EU) 19.5 38.9 — Ref-2 RMP (US)20.2 39.8 —

Conclusion of Screening Experiment 1

The results obtained from Bioanalyzer and DSF testing were combinatelyevaluated by means of the ANOVA model for response surfaces in order todetermine the best compositions that can possibly guarantee the highestthermal stability to the protein.

The list of the compositions recommended are reported in Table 12, whichalso compares the performances of the resulting prototypes formulationswith the Humira RMP, in terms of unfolding temperature and fragmentationchange over 1 month at 40° C.

Formulation C corresponds to DoE1 Formulation 25 and the real data werereported.

Comparing these formulas to the RMP it can be concluded that thebehavior of these prototype formulations in response to thermal stressis comparable with that observed for the RMP.

TABLE 12 Outcome of DoE1 experiments: recommended compositions forsecond screen Salt (NaCl) Buffer type Form mM (10 mM) pH Stabilizer C100 Histidine 6.4 Trehalose dihydrate (200 mM)

Somewhat unexpectedly, formulations containing trehalose dihydrate asthe sole stabilizer performed extremely well, especially in terms offragmentation inhibition, unfolding inhibition, and pH maintenance. Suchtrehalose-based formulations also exhibited good performance in terms ofaggregation and precipitation. That trehalose was such a strongcandidate as a stabiliser, especially on its own, was extremelypromising in view of its antioxidant properties, which would impartfurther long-term chemical stability (especially vis a vis oxidationand/or photo-oxidation) to adalimumab formulations. Furthermore, thattrehalose can be used alone and yet still exhibit excellent performance,was considered especially encouraging and paved the way to less complexformulations employing fewer components—this would in turn reduceprocessing and costs associated with the production of the relevantadalimumab drug product. As such, these trehalose-based formulationswere taken into a second round of screening experiments in order tofine-tune the formulations.

Screening Experiment 2—Analysis and Screening of Example 2 FormulationsAgainst Comparative Formulations of Example 3

A formulation prototype from the previous screen was identified (Table12). Since the previous step was conducted with no surfactant added, thesecond step aimed to screen a series of levels of compounded Polysorbate80 surfactant (range: 0-1 mg/mL) in order to assess whether surfactantaddition is required to favor protein stability.

Table 3 (Example 2) summarizes the design of this second step of thestudy and lists the formulations (DoE2 formulations) tested in thissecond screening exercise.

Typically, surfactants have been observed to contrast mechanicalstress-induced aggregation and shaking stress tests have been thereforeexecuted so as to evaluate how Polysorbate 80 affects protein stabilityand response to shaking.

As with Step 1, the reference compositions described in Example 3 havealso been evaluated so as to provide a baseline for the development of anew formulation.

The complete list of analyses conducted on this block of formulations isreported in Table 13. In this second screen, the respective formulationswere exposed to three different types of stress, thermal, mechanical,and light.

TABLE 13 Panel of analytical tests carried out on DoE2 formulations(Step 2): 1-month thermal stress conditions at 40° C. (A), shakingstress at 200 rpm (B) and light exposure according to ICH Q1B (C). A.Thermal stress at 40° C. Accelerated (40° C.) Stability time (weeks)Methods Test 0 2 w 4 w OD Content x — x iCE280 Isoforms x x x SE-HPLCAggregates x x x Bionalyzer Purity x x x pH pH x x x OsmolalityOsmolality x — — Nephelometry Turbidity x x x DSF Unfolding T x — — B.Shaking stress conditions Shaking stress (200 rpm) Stability time(hours) Methods Test 0 24 h 48 h OD Content x — — SE-HPLC Aggregates x xx Bioanalyzer Purity x x x pH pH x x x Nephelometry Turbidity x x x C.Light Exposure 7 hours of exposure at 765 W/m² (ICH Q1B). Light exposureSample Methods Test Time 0 Exposed OD Content x — iCE280 Isoforms x xSE-HPLC Aggregates x x Bioanalyzer Purity x x pH pH x x NephelometryTurbidity x x

Thermal stress tests were performed by simply heating a sample of therelevant formulations at the stipulated temperature for the stipulatedamount of time (typically 2 weeks or 4 weeks/1 month).

Mechanical stress tests were performed by simply mechanically shaking asample of the relevant formulations at room temperature at 200 rpm forthe stipulated period of time (typically 24 hours or 48 hours).

Light stress tests were performed by simply exposing a sample of therelevant formulations to 765 W/m² light (in accordance with ICH Q1Bguidelines of the European Medicines Agency in relation tiophotostability testing of new active substances and medicinal products)for 7 hours.

2.1 Osmolality

The osmolality of the DoE2 screening formulations are reported in Table14. The values, comprised in the range 378-401 mOsm/kg are probablyoverestimated due to the presence of Trehalose dihydrate that can leadto some increase in viscosity affecting the cryoscopic point of thesolutions and hence the osmolality. This was confirmed by measurementsin relation to other test formulations, which were 3-fold diluted withWFI prior to the osmolality test in order to decrease the viscosity: thereal osmolality of all these formulas is <350 mOsm/kg.

TABLE 14 Osmolality of DoE2 screening formulations (tested undiluted)Surfactant Salt (NaCl) (Polysorbate 80) Form concentration Buffer typeconcentration # (mM) (10 mM) pH Stabilizer (mg/mL) Time 0 DoE2-7 50Histidine 6.4 Trehalose dihydrate (200 mM) 0 381 DoE2-8 50 Histidine 6.4Trehalose dihydrate (200 mM) 0.5 381 DoE2-9 50 Histidine 6.4 Trehalosedihydrate (200 mM) 1 378

2.2 Protein Content (OD)

The protein content of all the DoE2 formulations at time 0 were in linewith the protein concentration target of 50 mg/mL (Table 15).

TABLE 15 Protein content (OD) of DoE2 screening formulations (testedundiluted) Surfactant Salt (NaCl) (Polysorbate 80) Form concentrationBuffer type concentration # (mM) (10 mM) pH Stabilizer (mg/mL) Time 0DoE2-7 50 Histidine 6.4 Trehalose dihydrate (200 mM) 0 49.9 DoE2-8 50Histidine 6.4 Trehalose dihydrate (200 mM) 0.5 50.2 DoE2-9 50 Histidine6.4 Trehalose dihydrate (200 mM) 1 50.42.3 Aggregates with Thermal Stress (SE-HPLC)

The variations in total aggregates by SE-HPLC are presented in FIG. 5.FIG. 5 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE2 formulations (of Example 2), along with referencestandards (representing comparator HUMIRA® formulations), at anarbitrary start point (red bars, time=0) and after both 2 weeks (greenbars) and 4 weeks (purple bars) of the formulation(s) being heated at40° C.

Minimal changes were observed for all the formulation, being the totalaggregates amount after 1 month at 40° C. below 1%.

The performances of the DoE2 screening formulations arecomparable/slightly better than those of the RMP materials.

2.4 Fragmentation with Thermal Stress (Bioanalyzer)

The variations in fragments by Bioanalyzer are presented in FIG. 6. FIG.6 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE2 formulations (of Example 2), along withreference standards (representing comparator HUMIRA® formulations), atan arbitrary start point (blue bars, time=0) and after both 2 weeks (redbars) and 4 weeks (green bars) of the formulation(s) being heated at 40°C.

Formulation DoE2-7 (no Polysorbate 80) undergoes a consistent increasein fragments whilst the other two, in presence of surfactant, were foundto be comparable to the RMP materials. Considering the data availablefrom the DoE1 experiments on formulation #25 (comparable to Form 7 ofthe DoE2), it can be concluded that the increased degradation of DoE2-7can be attributed to a possible contamination of the sample.

2.5 Isoforms Profile with Thermal Stress (iCE280)

The main peak and acidic cluster changes of the three formulations over1 month at 40° C. are reported in FIGS. 7 and 8 respectively

FIG. 7 is a bar chart showing the main peak isoforms profile, asdetermined by iCE280 analysis, of the DoE2 formulations (of Example 2)at an arbitrary start point (blue bars, time=0) and after both 2 weeks(red bars) and 4 weeks (green bars) of the formulation(s) being heatedat 40° C.

FIG. 8 is a bar chart showing the acid cluster peak(s) isoforms profile,as determined by iCE280 analysis, of the DoE2 formulations (of Example2) at an arbitrary start point (blue bars, time=0) and after both 2weeks (red bars) and 4 weeks (green bars) of the formulation(s) beingheated at 40° C.

The highest changes are observed in DoE2-7 (−15% in the main peak), butthis may derive from a possible contamination of the sample, aspreviously highlighted.

These results confirm the experimental evidences already highlighted byiCE280 on the prototype formulations (resulting from the firstscreening): formulations in histidine present comparable degradationrates in terms of isoforms profile to RMP.

The results, in terms of acidic cluster, are in line with theobservations made for the main peak.

2.6 pH Screen with Thermal Stress

The variation in pH of the DoE2 formulations (of Example 2) over aperiod of time during which the formulations are heated at 40° C. isshown in Table 16.

pH decreases were observed in DoE2-7, as shown in Table 16. This mayderive from possible contaminations/bacteria proliferation in thesamples.

TABLE 16 DoE2 screening: pH (thermal stress at 40° C.) Surfactant Salt(NaCl) (Polysorbate 80) Form concentration Buffer type concentration 2weeks 4 weeks # (mM) (10 mM) pH Stabilizer (mg/mL) Time 0 (40° C.) (40°C.) DoE2-7 50 Histidine 6.4 Trehalose dihydrate (200 mM) 0 6.4 4.3 4.3DoE2-8 50 Histidine 6.4 Trehalose dihydrate (200 mM) 0.5 6.4 6.4 6.4DoE2-9 50 Histidine 6.4 Trehalose dihydrate (200 mM) 1 6.4 6.4 6.42.7 Turbidity with Thermal Stress (Nephelometry)

FIG. 9 is a bar chart showing the turbidity, as determined byNephelometry, of the DoE2 formulations (of Example 2) at an arbitrarystart point (blue bars, time=0) and after both 2 weeks (red bars) and 4weeks (green bars) of the formulation(s) being heated at 40° C.

The turbidity of the three formulations is, time 0, in the range oftypically opalescent solutions (6-18 NTU). With respect to theoriginating DS materials, of typical turbidity of 19-52 NTU, the DPsolutions after aseptic filtration are considerably clarified.

Importantly, turbidity values of Humira RMP are normally around 10 NTU,in line with our formulas.

2.8 Aggregates with Mechanical Stress (SE-HPLC)

FIG. 10 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE2 formulations (of Example 2) at an arbitrary startpoint (blue bars, time=0) and after both 24 hours (red bars) and 48hours (green bars) of the formulation(s) being mechanically agitated(shaking).

The variations in total aggregates by SE-HPLC are presented in FIG. 10.

Minimal changes (+0.1%) were observed for all the formulations inhistidine buffer.

2.9 Fragmentation with Mechanical Stress (Bioanalyzer)

FIG. 11 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE2 formulations (of Example 2) at an arbitrarystart point (blue bars, time=0) and after both 24 hours (red bars) and48 hours (green bars) of the formulation(s) being mechanically agitated(shaking).

The variations in fragments by Bioanalyzer are presented in FIG. 11.Minimal changes are observed, being all the values recorded equal to orlower than 0.5%.

After 48 hour shaking at room temperature all the samples presentedfragmentation in the range 0.2-0.4%. No trend towards fragmentationincreases was highlighted upon mechanical shaking.

2.10 pH Screening with Mechanical Stress

The variation in pH of the DoE2 formulations (of Example 2) over aperiod of time during which the formulations are mechanically agitated(shaking) is shown in Table

17. No changes where observed.

TABLE 17 DoE2 screening: pH (mechanical shaking) Surfactant Salt (NaCl)(Polysorbate 80) Form concentration Buffer type concentration # (mM) (10mM) pH Stabilizer (mg/mL) Time 0 24 hours 48 hours DoE2-7 50 Histidine6.4 Trehalose dihydrate (200 mM) 0 6.4 6.5 6.5 DoE2-8 50 Histidine 6.4Trehalose dihydrate (200 mM) 0.5 6.4 6.4 6.4 DoE2-9 50 Histidine 6.4Trehalose dihydrate (200 mM) 1 6.4 6.4 6.42.11 Turbidity with Mechanical Stress (Nephelometry)

FIG. 12 is a bar chart showing the turbidity, as determined byNephelometry, of the DoE2 formulations (of Example 2) at an arbitrarystart point (blue bars, time=0) and after both 24 hours (red bars) and48 hours (green bars) of the formulation(s) being mechanically agitated(shaking). No changes were observed.

2.12 Aggregates with Light Stress (SE-HPLC)

FIG. 13 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE2 formulations (of Example 2), along with referencestandards (representing comparator HUMIRA® formulations), beforeexposure to light (blue bars, time=0) and after 7-hour light exposure at765 W/m² (red bars).

Comparisons were also made with Humira samples (from US and EU) treatedat the same conditions. In the RMP, aggregation increases up to 9-15%upon light exposure (at time 0 aggregates are lower than 1%). All theDoE2 formulations present lower or comparable increases and thereforebetter/similar resistance to thermal stress. More in detail:

-   -   Formulations in histidine buffer: 5.8→9.2% total aggregates upon        light exposure        2.13 Fragmentation with Light Stress (Bioanalyzer)

FIG. 14 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE2 formulations (of Example 2), along withreference standards (representing comparator HUMIRA® formulations),before exposure to light (blue bars, time=0) and after 7-hour lightexposure at 765 W/m² (red bars).

Minimal increases were highlighted (+0.3% at most, after exposure). Allfragmentation amounts are well below 1% after 7-hour exposure (FIG. 14).

2.14 Isoforms Profile with Light Stress (iCE2280)

FIG. 15 is a bar chart showing the main peak isoforms profile, asdetermined by iCE280 analysis, of the DoE2 formulations (of Example 2),along with reference standards (representing comparator HUMIRA®formulations), before exposure to light (blue bars, time=0) and after7-hour light exposure at 765 W/m² (red bars).

FIG. 16 is a bar chart showing the acid cluster peak(s) isoformsprofile, as determined by iCE280 analysis, of the DoE2 formulations (ofExample 2), along with reference standards (representing comparatorHUMIRA® formulations), before exposure to light (blue bars, time=0) andafter 7-hour light exposure at 765 W/m² (red bars).

In the Humira RMP, the light exposure determines significant effects:most relevantly, decreases in main peak abundance (around −9%) andconcurrent increase in acidic cluster (up to +15%), related tophotoxidation phenomena, are observed.

Formulas in histidine were found to be more susceptible to degradationresulting from light exposure than the RMP: decreases in main peakabundance are −11.4% (DoE2-7) or even more (around −18% for the others),increases in acidic cluster amounted to up to +27%.

Histidine is susceptible to oxidation deriving from both extensive lightexposure and degradation products (typically peroxides) released bypolysorbates under stressing conditions. Therefore, polysorbate80+histidine is a combination which may create increased instabilityunder light stress.

In order to better elucidate the impact of surfactant and determinewhether it is required to prevent protein degradation/particle formationupon freeze-thawing cycles, dedicated experiments were performed whichhighlighted that no added value is given by Polysorbate 80. This couldeventually lead to a surfactant-free back up formula in histidine.

2.15 Turbity with Light Stress (Nephelometry)

FIG. 17 is a bar chart showing the turbidity, as determined byNephelometry, of the DoE2 formulations (of Example 2) before exposure tolight (blue bars, time=0) and after 7-hour light exposure at 765 W/m²(red bars). Essentially no changes were observed.

2.16 pH Screen with Light Stress

The variation in pH of the DoE2 formulations (of Example 2), over aperiod of time during which the formulations are exposed for 7-hours tolight at 765 W/m², is shown in Table 18. No changes where observed.

TABLE 18 DoE2 screening: pH (light exposure) Surfactant Salt (NaCl)(Polysorbate 80) Form concentration Buffer type concentration After #(mM) (10 mM) pH Stabilizer (mg/mL) Time 0 exposure DoE2-7 50 Histidine6.4 Trehalose dihydrate (200 mM) 0 6.4 6.5 DoE2-8 50 Histidine 6.4Trehalose dihydrate (200 mM) 0.5 6.4 6.5 DoE2-9 50 Histidine 6.4Trehalose dihydrate (200 mM) 1 6.4 6.5

2.17 Effect of Surfactant on Freeze-Thawing Cycles

Isoforms profiles, aggregates and sub-visible particles of the threeDoE2 formulations have been determined before and after fivefreeze-thawing cycles (−80° C.→room temperature) in order to assesswhether the surfactant exerts any impact.

FIG. 18 is a bar chart showing the main peak isoforms profile, asdetermined by iCE280 analysis, of the DoE2 formulations (of Example 2)before (blue bars, time=0) and after m² (red bars) five freeze-thawingcycles (−80° C.→room temperature).

FIG. 19 is a bar chart showing the acid cluster peak(s) isoformsprofile, as determined by iCE280 analysis, of the DoE2 formulations (ofExample 2) before (blue bars, time=0) and after m² (red bars) fivefreeze-thawing cycles (−80° C.→room temperature).

FIG. 20 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE2 formulations (of Example 2), along with referencestandards (representing comparator HUMIRA® formulations) before (bluebars, time=0) and after m² (red bars) five freeze-thawing cycles (−80°C.→room temperature).

FIG. 21 is a bar chart showing the number concentration (#/mg) ofsub-visible particles with a particle size less than or equal to 10microns, as determined by sub-visible particle count analysis, of theDoE2 formulations (of Example 2) before (blue bars, time=0) and after m²(red bars) five freeze-thawing cycles (−80° C.→room temperature).

FIG. 22 is a bar chart showing the number concentration (#/mg) ofsub-visible particles with a particle size less than or equal to 25microns, as determined by sub-visible particle count analysis, of theDoE2 formulations (of Example 2) before (blue bars, time=0) and after m²(red bars) five freeze-thawing cycles (−80° C.→room temperature).

No changes in isoforms and in aggregates were observed (FIGS. 18-20)upon freeze-thawing, whilst minimal, non critical changes (FIGS. 21-22)in sub-visible particles were highlighted, and found to be non-relatedto the presence of surfactant. Higher particles count in DoE2-8 to bemost likely related to sample manufacturing.

Therefore, there is no added value in adding a surfactant with the aimof preventing particles and aggregates formation/protein degradation inthe course of freeze-thawing cycles. This highlights the effectivenessof the novel formulations irrespective of surfactant.

Conclusion of Screening Experiment 2

On the basis of the data collected, relevant to thermal, mechanical andlight stress, the following conclusion can be made:

Formulations in 10 mM Histidine Buffer at pH 6.4 (DoE2-7, DoE2-8,DoE2-9):

-   -   Upon thermal stress, performances comparable to Humira were        highlighted    -   Minimal increase in aggregation upon mechanical shaking.    -   Increased degradation and isoforms profile change with respect        to Humira due to susceptibility of histidine to light and        degradation products from Polysorbate 80. The formulation        without Polysorbate 80 in this group (Doe2-7) is still slightly        worse than RMP, but remarkably better than the others in        histidine+Polysorbate 80 (0.5 or 1.0 mg/mL).

The presence of Polysorbate 80 has been evaluated to assess itseffectiveness and function as a protectant for the protein (protectionagainst freeze-thawing). Upon 5× freeze-thawing cycles (−80° C.→roomtemperature) it was observed that no added value is given by thesurfactant, and the recommendation is to further progress DoE2-7 whichis surfactant-free (50 mg/mL Adalimumab, 200 mM Trehalose dihydrate, 50mM sodium chloride in 10 mM histidine pH 6.4).

Based on the screening work carried out on different formulationsvarying in buffer/pH, stabilizer, isotonicity agent (NaCl) amount andsurfactant (Polysorbate 80) level, the best composition, showingcomparable or even improved characteristics with respect to Humira upondifferent stressing conditions (thermal, mechanical, light) has beenidentified as:

Ingredient Amount (mg/mL) Adalimumab 50    Histidine (anhydrous) 1.55 *Trehalose dihydrate 75.67 ** Sodium chloride  2.92 *** WFI and sodiumhydroxide q.b. to adjust pH to 6.4 * corresponding to 10 mM histidine;** corresponding to 200 mM; *** corresponding to 50 mM

Such formulations can be readily incorporated within pre-filled glasssyringes with 29G ½″ needles).

Abbreviations

DoE Design of experimentDP Drug productDS Drug substanceDSF Differential scanning fluorimetryOD Optical density

PES Polyethersulphone

rpm rounds per minute

RT Room Temperature

SE-HPLC Size exclusion high performance liquid chromatographySMI Summary manufacturing instructionsSOP Standard operating procedureWI Working instruction

1. A liquid pharmaceutical composition comprising: (a) adalimumab; (b)an histidine buffering agent (or histidine buffer system); and (c) asugar stabiliser; wherein the composition has a pH greater than or equalto pH 6.30.
 2. The liquid pharmaceutical composition as claimed in claim1, wherein the composition has a pH between 6.3 and 6.5.
 3. The liquidpharmaceutical composition as claimed in any preceding claim, whereinthe sugar stabiliser is trehalose.
 4. The liquid pharmaceuticalcomposition as claimed in any preceding claim, wherein the compositionis either (substantially or entirely) free of amino acids other thanhistidine or comprises one or more amino acids other than histidine in a(collective) concentration of at most 0.1 mM.
 5. The liquidpharmaceutical composition as claimed in claim 4, wherein thecomposition is free of amino acids other than histidine.
 6. The liquidpharmaceutical composition as claimed in any preceding claim, whereinthe composition is either (substantially or entirely) free ofsurfactants or comprises one or more surfactants in a (collective)concentration of at most 0.001 mM.
 7. The liquid pharmaceuticalcomposition as claimed in any preceding claim, wherein the compositionis either (substantially or entirely) free of arginine (suitablyL-arginine) or comprises arginine in a concentration of at most 0.1 mM.8. The liquid pharmaceutical composition as claimed in any precedingclaim, wherein the composition is either (substantially or entirely)free of phosphate buffering agents or comprises a phosphate buffersystem in a concentration of at most 0.1 mM.
 9. The liquidpharmaceutical composition as claimed in any preceding claim, furthercomprising a tonicifier selected from sodium chloride, potassiumchloride, magnesium chloride, or calcium chloride.
 10. The liquidpharmaceutical composition as claimed in any preceding claim, whereinthe osmolality of the composition is between 220 and 390 mOsm/kg. 11.The liquid pharmaceutical composition as claimed in any preceding claim,wherein the protein unfolding temperature of adalimumab in the liquidpharmaceutical composition is greater than or equal to 70° C.
 12. Theliquid pharmaceutical composition as claimed in any preceding claim,wherein the composition comprises adalimumab, histidine (or histidinebuffering species), and trehalose in a weight ratio of25-75:0.31-7.8:15-140 respectively.
 13. The liquid pharmaceuticalcomposition as claimed in any preceding claim, wherein the compositioncomprises adalimumab, histidine (or histidine buffering species),trehalose, and sodium chloride in a weight ratio of45-55:0.77-2.2:65-72:2.7-3.1 respectively.
 14. The liquid pharmaceuticalcomposition as claimed in any preceding claim, wherein the compositioncomprises: 45 to about 55 mg/ml adalimumab; 5 to 14 mM histidine (orhistidine buffer system); 190 to 210 mM trehalose; 40 to 60 mM sodiumchloride; water (for injection); wherein the composition: has a pHbetween 6.3 and 6.5; is free of arginine or comprises arginine in aconcentration of at most 0.001 mM; is free of amino acids other thanhistidine or comprises one or more amino acids other than histidine in a(collective) concentration of at most 0.001 mM. is free of surfactantsor comprises one or more surfactants in a (collective) concentration ofat most 0.0001 mM; and/or is free of phosphate buffering agents (e.g.sodium dihydrogen phosphate, disodium hydrogen phosphate) or comprises aphosphate buffer system in a concentration of at most 0.001 mM.
 15. Adrug delivery device comprising a liquid pharmaceutical composition asclaimed in any preceding claim.
 16. A liquid pharmaceutical compositionas claimed in any of claims 1 to 14 for use in the treatment ofrheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis,Crohn's disease, ulcerative colitis, moderate to severe chronicpsoriasis and/or juvenile idiopathic arthritis.